mpsa abstracts
TRANSCRIPT
s of Protein Chemistry, Vol. J3, No. 5, July t994
l Oth International Conference on Methods in Protein Structure Analysis (September 8-13, 1994, Snowbird, Utah)
ABSTRACTS
Special Issue Editors: M. Zouhair Atassi Ettore Appella
515
0027-8033/94/0700 0515507.00/0 �9 1994 Plenum Publishing Corporation
Journal of Protein Chemistry, Vol. 13, No. 5, July 1994
MPSA Abstract Listing
A1. Mahmoud Aminlari A2. Thomas Asquith and
Katherine Sarlo B1. Jerome M. Bailey, Oanh Tu,
Gilbert Issai, Alice Ha, and John E. Shively
B2. Alexander W. Bell, Nicole C. Baur, John J. M. Bergeron, Wei-Jia Ou, David Y. Thomas, Katherine Cianflone, Allain Baldo, Maxwell T. Hincke, Richard L. Momparler, Josde Lalibert6, David M. P. Thomson, and M. Sutherland
B3. Vladimir Besada, Javier Gonzalez, Gabriel Padron, Hilda Garay, Osvaldo Reyes, Toshifumi Takao, and Yasutsugu Shimonishi
B4. Rainer Bischoff, Dominique Roecklin, Bernadette Bouchon, Klaus Klarskov, and Alain Van Dorsselaer
B5. Patricia G. Brake, Anne Pacitti, Terry Higgins, Panos Stevis, John Malinowski, Sue McElhiney, James Huang, and Christine Vestal
B6. John M. Brewer, Vani S. Sangadala, Robert L. Robson, Claiborne C. V. Glover, Michael J. Holland, and Lukasz Lebioda
B7. Scott D. Buckel, Tracy Stevenson, and Joseph A. Loo
C1. Martin Caffrey, Jin Wang, Carmichael J. A. Wallace, and Ian Clark-Lewis
C2. C. A. Carothers Carraway, J. Huang, Y. Li, S.-H. Juang, A. Gallo, B. J. Mayer, and K. L. Carraway
Probing the active site of rhodanase with disulfide reagents Uses of gel electrophoresis to monitor detergent enzymes: application to safety assessments C-terminal sequence analysis of polypeptides containing C-terminal proline
Exploiting automated protein sequence analysis: in situ cleavage and resequencing
Study of a rearrangement at the C-terminus of peptides during the collision activated dissociation experiments
Analysis of the microheterogeneity of recombinant Schistosoma mansoni antigen rSmp28 reveals N-formyl-alanine as a new post-translational modification in Saccharomyces cerevisiae
Characterization of a recombinant cytosolic domain of CD45 phosphatase
Preparation and characterization of the site-directed E211Q mutant of yeast enolase 1
Direct mass spectrometric analyses for protein chemistry studies
Structure characterization of membrane bound and surface adsorbed protein
p58, A membrane-and microfilament-associated gag-like protein from ascites tumor cell microvilli, binds SrC SH3 domain through a poly-proline motif
517 0027-8033/94/0700-0517507.00/0 �9 1994 Plenum Publishing Corporation
518 MPSA Abstract Listing
C3. Patrick L. Coleman and Daniel Sarpong
D1. David W. Deerfield, Amanda Holland-Minkley, John D. Hempel, and Hugh B. Nicholas, Jr.
D2. Nancy D. Denslow, Leroy C. Folmar, and Craig V. Sullivan
D3. James D. Dixon, Jonathan P. Mark, Christopher P. Elicone, Simin D. Maleknia, Brian F. McGuinness, Fred. E. Regnier, and Noubar B. Afeyan
D4. Julia M. Dolence and C. Dale Poulter
El. Tsezi Egorov, Alexander Musolyamov, Yves Popineau, Jens Andersen, and Peter Roepstorff
F1. Roberto J. Falkenstein, Mirtha J. Biscoglio de Jimenez Bonino, and Clara Pefia
G1. D. L. Gauggel, T. N. Asquith, R. J. Isfort, N. S. Miller, and D. B. Cody
G2. Michael F. Giblin, Tuck C. Wong, and Thomas P. Quinn
G3. Gregory A. Grant and Mark W. Crankshaw
G4. Scott Griffith, Steve Schroeder, and Thomas Quinn
G5. F. Guinet, Y. Petillot, J. M. Chapsal, J. Dubayle, F. Greco, O. Barge, E. Forest, and C. Valentin
H1. Frederick M. Hahn, Jonathan A. Baker, and C. Dale Poulter
H2. Torben Halkier and Arne Agerlin Olsen
H3. Mitsuru Haniu, William C. Kenney, and Michael F. Rohde
H4. James G. Harman, Eun Ju Lee, Joel Glasgow, Sew Fen Lew, and Ali O. Belduz
Direct determination of immobilized protein concentration
Conformational flexibility of the Gly-Gly dipeptide within protein structures
A highly conserved N-terminal sequence for teleost vitellogenins
Rapid fingerprinting of proteins with immobilized protease columns
Protein, farnesyi transferase. Evidence for an electrophilic substitution mechanism. Disulphide mapping of prolamins
Conformational comparison in the snake toxin family
Mapping and characterization of proteins associated with morphological transformation in the SHE cell assay
Solution structure of a cyclic c~-MSH analogue--initial NMR studies
Identification of modified PTH-amino acids in protein sequence analysis Crystallization of a thermal stable trypsin inhibitor from Phaseolus lunatus
Characterization of recombinant porin by mass spectrometry
Insights into the structure and active-site architecture of IPP:DMAPP isomerase
Counting cysteine and cystine residues in proteins and peptides using MALDI-TOF mass spectrometry Detection of di-PTH-cys for assignment of disulfide linkages
Mutagenesis of the cyclic AMP receptor protein (CRP) of Escherichia coli
MPSA Abstract Listing 519
Reed J. Harris H5.
H6. Reed J. Harris, Michael S. Molony, Lene H. Keyt, and Shiaw-Lin Wu
H7. David H. Hawke, Jacqueline Tso, Sherrell Early, and Chad Miller
H8. G. Thomas Hayman and Jan A. Miernyk
H9. Ulf Hellman, Christer Wernstedt, and Jorge Gdfiez
H10. Daniel Hess, Ralph Studer, and Peter E. Hunziker
Hl l . Hisashi Hirano, Yoshihiro Watanabe, Sergei F. Barbashov, Setsuko Komatsu, Andrew M. Hemmings, Masaru Miyagi, and Susumu Tsunasawa
H12. Reuben E. Huber, Nathan J. Roth, and Michael T. Gaunt
J1. Paul JeniS, Thierry Mini, Suzette Moes, and Martin Horst
J2. Kenji Jinnai, Tetsuo Ashizawa, and M. Zouhair Atassi
J3. Anders H. Johnsen, Hanne Jensen, and Jens F. Rehfeld
K1. Masaharu Kamo, Takao Kawakami, Norifumi Miyatake, and Akira Tsugita
K2. J. N. Keen, P. F. Zagalsky, and J. B. C. Findlay
K3. Regine Kraft, Susanne Kostka, and Enno Hartmann
K4. Henry C. Krutzsch and John K. Inman
M1. Claudia Machalinski and Mirtha Biscoglio de Jimdnez Bonino
M2. Donald K. McRorie, Gregg R. Dieckmann, Susan Heilman, William F. DeGrado, and Vincent L. Pecoraro
M3. Chad G. Miller, James Kenny, Julie Sahakian, David H. Hawke, and Jacqueline Tso
Evaluating protein modification sites observed by N-terminal sequence analysis Detection of low levels of hydroxylysine in non-collagenous proteins
Chemical examination of the diphenylphosphoroisothiocyanatidate/trimethylsilnolate method for protein carboxyl-terminal degradations Aptase and molecular chaperone activities of the maize endoplasmic reticulum-resident Stress-70 protein Micropreparation for sequence analysis of peptides of proteins obtained from old archived polyacrylamide gels by in situ trypsin digestion Identification and characterization of metal protein complexes of crude cell extracts by mass-spectrometry The structure and function of plant leginsulin
Probing the Mg 2+ binding site of/3-galactosidase (E. coli)
In-gel reduction and alkylation of proteins and enzymatic digestion: application to the sequence analysis of proteins separated by 1-D gel electrophoresis Analysis of the extracellular organization of the u-chain of mouse acetylcholine receptor on live muscle cells
Probing of the phylogeny of the CCK/gastrin family by antisera directed against the conserved common C-terminus Separation and characterization of proieins with two electrophoresis
An investigation of the bathochromic shift phenomenon in biology
Microsequencing of proteins of the endoplasmic reticulum (ER) membrane N-isopropyliodoacetamide in the reduction and alkylation of proteins: Use in microsequence analysis Determination of the primary structure of a new milk-clotting protease by microsequencing techniques
A pH dependence on the association properties of model coiled coil peptides
C-terminal protein sequence analysis using the Hewlett-Packard C-terminal protein sequencing system
520 MPSA Abstract Listing
M4. Jeffrey A. Moore and C. Dale Poulter
M5. Mary B. Moyer and William A. Burkhart
M6. Tatyana Muranova and Lubov Makova
N1. Hugh Nicholas, John Hempel, Amy Hinich, David Deerfield, Joseph Behrmann, and Alex Ropelewski
N2. Lori Nixon and Leonard Maneri
N3. Kerry Nugent and Ken Stoney
N4. Kerry Nugent and John Wieser
O1. Hiroshi Ohguro, Krzysztof Palczewski, Kenneth A. Walsh, and Richard S. Johnson
P1. Leonard C. Packman, Carl Webster, and John Gray
P2. G. Padr6n, V. Morera, L. J. Gonzfilez, Y. T~mbara, V. Besada, R. Villalonga, G. Chinea, O. Reyes, H. Garay, R. Bringas, and C. Nazfibal
P3. Bruce P. Parkinson, Kent A. Yamada and Anne Randolph
P4. Anthony Pisano, Nicole H. Packer, John W. Redmond, Keith L. Williams, and Andrew A. Gooley
R1. Hanne H. Rasmussen, Ejvind MCrtz, Matthias Mann, Peter Roepstorff, and Julio E. Celis
R2. Lone K. Rasmussen, Esben S. SCrensen, Torben E. Petersen, Jcrgen Gliemann, and Poul Henning Jensen
R3. Staffan Renlund, Henrik Wadensten, Annika Persson, Per Persson, Agneta Johansson, and Per-Olof Edlund
R4. Donald J. Rose
S1. Ragna Sack and Peter E. Hunziker
Escherichia coli DMAPP-tRNA transferase expression and characterization Alternative reverse-phase supports for direct electroelution and in situ digestion on the Hewlett-Packard sequencing column A convenient procedure for determination of carboxyterminal amino acids of proteins immobilized on the membrane Quantitative evaluation of different multiple alignment programs
Degradative modifications of rhIL-lra (recombinant human interleukin receptor antagonist) Automated preparation of complex biological samples prior to protein or peptide sequence analysis A new PTH amino acid separation system to improve performance at the low picomole level for older sequencers Arrestin topographic study using selective chemical modifications and hydrogen-deuterium exchange analyzed by mass spectrometry
The use of maldron mass spectrometry and amino acid sequence analysis in characterizing small amounts on N-blocked protein Characterization of antigenic determinants of P64K a Neisseria rneningitidis membrane protein
A straightforward method for amino-terminal sequencing of gamma-carboxyglutamic acid using standard Edman chemistry
Characterization of individual N- and O- linked glycosylation sites using Edman degradation
Identification of proteins recorded in human 2-D gel protein databases by mass spectrometric peptide mapping
Localization of transglutaminase reactive glutamine and lysine residues in Alzheimer amyloid/3A4 peptide: Cross-linking to extracellular matrix proteins and c~2M receptor
Different forms of apolipoprotein A and alcohol dehydrogenase studied by electrospray LC/MS
Automated two-dimensional electrophoresis-liquid chromatography for the micropreparative isolation of proteins Determination of cysteine by amino acid analysis: Online oxidation and hydrolysis
MPSA Abstract Listing 521
$2. Julie Sahakian, Alex Apffel, Chad Miller, and Rodney L. Levine
$3. Kazuyasu Sakaguchi, Nicola Zambrano, Marc S. Lewis, Eric T. Baldwin, Bruce A. Shapiro, John W. Erickson, James G. Omichinski, G. Marius Clore, Angela M. Gronenborn, and Ettore Appella
$4. Werner Schr/Sder and Irmgard Moser
$5. Werner Schr/3der, Werner Pansegrau, and Erich Lanka
$6. Richard J. Simpson, James Eddes, Hong Ji, Gavin E. Reid, and Robert L. Moritz
$7. Esben S. SCrensen, Lone K. Rasmussen, Poul Henning Jensen, Peter Hcjrup, and Torben E. Petersen
$8. David W. Speicher, David F. Reim, and Kaye D. Speicher
$9. B. R. Srinivasa and S. P. Barde
$10. William G. Stirtan and C. Dale Poulter
Sll. Alyona Sukhanova, Sergey Vorob'ev, Alexander Gabibov, and Igor Bronstein
T1. Kenji Tanaka, Kuniko Einaga, Minoru Tsukada, Jonathan F. Tait, and Kazuo Fujikawa
T2. Akira Tsugita, Masaharu Kamo, Keiji Takamoto, and Kazuo Satake
V1. Ilya A. Vakser V2. V. V. Velikodvorskaia, A.
G. Gabibov, and A. G. Rabinkov
V3. Tennie Videler, Michael Osborne, Geoffrey Moore, Richard James, and Colin Kleanthous
V4. Nikolie Vtyurin W1. Jane H. Walent, Richard
Bessen, Dick Marsh, and Ronald L. Niece
Identification of an oxidative modification of glutamine synthetase using electrospray LC/MS and Edman sequencing techniques
Characterization of a binding site of the human immunodeficiency virus type 1 RNA for the nucleocapsid protein P7
Functional and protein analytical studies on the major outer membrane protein of Campylobacter jejuni Relaxase (TraI) of IncP o~ Plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA High-speed chromatographic separation of proteins and peptides: Application to rapid peptide mapping of in-gel digested proteins
Identification of posttranslational modifications in bovine osteopontin: localization of 28 phosphorylation sites, 30-glycosylation sites and 2 transglutaminase reactive glutamines
Integration of MALDI mass spectrometry with in situ protease digestions on high retention PVDF membranes to obtain internal sequences at maximum sensitivity A thiol mediated autolytic cleavage of homocysteinyl prolyl bond
Characterization of purified yeast protein geranylgeranyltransferase type I using a continuous fluorescence assay The DNA-topoisomerase I. Analysis by limited proteolysis of domain structure and conformational changes
Preparation and characterization of a conjugate of annexin V and the B-chain of urokinase
A novel C-terminal sequencing method using perfluoroacyl anhydrides
Protein docking in the absence of detailed molecular structures Acetyl-CoA-carboxylase: registration of active polymer complex
Structure determination by NMR of the nuclease inhibitor protein IM8
Local tight packing of hydrophobic groups in beta-structure Identification of distinct proteinase K cleavage sites in two strains of scrapie PRP by chemical isolation of unique sequence by orthophtalaldehyde and by comparison of N-terminal sequence maps of PRP fragments
52Z MPSA Abstract Listing
W2. Jane H. Walent, Francis H. C. Tsao, and Ronald L. Niece
W3. Hong Wang and C. Dale Poulter
W4. Scot R. Weinberger, Lynn M. Chakel, Alex Apffel, Julie Sahakian, and Chad G. Miller
W5. Ewald M. W0ndrak, Alan R. Kimmel, and John M. Louis
Identification of a cleavage-sensitive region in lipocortin 1 by isolation of unique N-terminal sequence by orthophtalaldehyde
Purification and general characterization of dimethylallyl tryptophan synthase A rapid screening strategy for the identification of modified peptide fragments of glutamine synthetase using the Hewlett-Packard MALDI-TOF system
Processing of the mature HIV-1 nucleocapsid protein by the viral protease
PR
OB
ING
TH
E A
CT
IVE
SIT
E O
F R
HO
DA
NE
SE
WIT
H D
ISU
LF
IDE
RE
AG
EN
TS
M
ahm
oud
Am
inla
ri,
Dep
artm
ent o
f B
ioch
emis
try,
Sch
ool o
f V
eter
inar
y M
edic
ine,
Shi
raz
Uni
vers
ity,
Shi
raz,
713
65
Iran
The
pur
pose
of
pres
ent i
nves
tiga
tion
was
to
obta
in a
n in
sigh
t int
o th
e ch
emic
al p
rope
rtie
s of
the
act
ive
site
of
the
enzy
me
rhod
anes
e (t
hios
ulfa
te:
cyan
ide
sulf
urtr
ansf
eras
e).
Thi
s en
zym
e is
wid
ely
dist
ribu
ted
in n
atur
e, f
rom
bac
teri
a to
man
. Se
vera
l fu
ncti
ons,
inc
ludi
ng
cyan
ide
deto
xifi
cati
on a
nd s
ynth
esis
of
iron
-sul
fur
cent
ers,
hav
e be
en p
ropo
sed
for
this
en
zym
e. D
iffe
rent
dis
ulfi
des
wit
h di
ffer
ent c
hem
ical
pro
pert
ies
wer
e al
low
ed to
rea
ct w
ith
the
sulf
hydr
yl g
roup
in t
he a
ctiv
e si
te o
f rho
dane
se. T
he E
llm
an r
eage
nt;
5,5'
-dit
hiob
is (
2-
nitr
oben
zoic
aci
d),
DT
NB
, re
acte
d ve
ry s
low
ly.
A m
arke
d in
crea
se in
the
rat
e of
rea
ctio
n of
rho
dane
se w
ith
DT
NB
was
obs
erve
d w
hen
cati
onic
dis
ulfi
des,
cys
tam
ine
and
cyst
ine,
w
ere
pres
ent.
No
chan
ge w
as s
een
wit
h no
n-ca
tion
ic d
isul
fide
s di
met
hyl
disu
lfid
e, 2
- hy
drox
y et
hyl d
isul
fide
, dit
hiog
lyco
lic
acid
oxi
dize
d gl
utat
hion
e, 2
,2'-
sali
cyli
c ac
id,
2,2'
- di
pyri
dyl
disu
lfid
e (2
-PD
S) a
nd 4
,4-D
ipyr
idyl
dis
ulfi
de (
4-PD
S).
A K
m o
f 2.
0 an
d 2.
5 m
mol
/L
was
ob
tain
ed
for
cyst
amin
e an
d cy
stin
e,
resp
ecti
vely
, w
hile
th
e K
m
for
thio
sulf
ate,
the
com
mon
sub
stra
te o
f th
e en
zym
e w
as 3
.0 m
mol
/L.
The
se r
esul
ts i
ndic
ate
exis
tenc
e of
ani
onic
gro
ups
in o
r in
the
vic
init
y of
the
acti
ve s
ite
whi
ch f
acil
itat
e bi
ndin
g of
the
cat
ioni
c di
sulf
ides
. Fu
rthe
rmor
e, t
hese
dat
a m
ight
sug
gest
tha
t th
ese
disu
lfid
e ar
e th
e tr
ue p
hysi
olog
ical
sub
stra
tes
of r
hoda
nese
.
Key
wor
ds:
Rho
dane
se, c
ysta
min
e, d
isul
fide
s.
Al
US
ES
OF
GE
L E
LE
CT
RO
PH
OR
ES
IS T
O M
ON
ITO
R D
ET
ER
GE
NT
EN
ZY
ME
S:
AP
PL
ICA
TIO
N T
O S
AF
ET
Y A
SS
ES
SM
EN
TS
.
Tho
mas
Asq
uith
and
Kat
heri
ne S
arlo
C
orpo
rate
Pro
fess
iona
l & R
egul
ator
y Se
rvic
es/H
uman
Saf
ety
Dep
t., T
he P
roct
er &
Gam
ble
Co.
, P.O
. B
ox 3
9870
7, C
inci
nnat
i, O
hio,
452
39-8
707.
Occ
upat
iona
l ex
posu
re g
uide
line
s ar
e on
e to
ol u
sed
to s
ucce
ssfu
lly
cont
rol a
ller
gy to
de
terg
ent e
nzym
es in
man
ufac
turi
ng f
acil
itie
s. T
hese
exp
osur
e gu
idel
ines
are
bas
ed o
n sa
fety
ass
essm
ents
whi
ch m
easu
re th
e al
lerg
enic
pot
enti
al o
f enz
ymes
. B
ecau
se p
rote
in
com
posi
tion
is o
ne f
acto
r tha
t det
erm
ines
all
erge
nic
pote
ntia
l, it
is im
port
ant t
hat
com
posi
tion
rem
ains
ess
enti
ally
unc
hang
ed a
fter
exp
osur
e gu
idel
ines
are
det
erm
ined
. G
el
elee
trop
hore
sis (
SD
S-P
AG
E a
nd I
EF)
pro
vide
s a
fast
and
cos
t eff
ecti
ve m
eans
to m
onit
or th
e co
mpo
siti
on o
f enz
ymes
. Si
gnif
ican
t cha
nges
in c
ompo
siti
on m
ay tr
igge
r ad
diti
onal
saf
ety
test
ing.
Gui
deli
nes
for
the
min
imum
acc
epta
ble
vari
abil
ity
are
no c
hang
es in
ele
ctro
phor
etic
m
obil
ity
or in
crea
se in
rel
ativ
e am
ount
gre
ater
than
10%
for
any
com
pone
nt c
ompr
isin
g 10
%
or g
reat
er o
f the
tota
l pro
tein
. T
hey
have
bee
n ap
plie
d to
mon
itor
sta
bili
ty o
f sk
in p
rick
test
re
agen
ts, t
o de
tect
con
tam
inan
ts, t
o as
sess
cha
nges
in p
rodu
ctio
n te
chni
ques
and
to q
uali
fy
new
sou
rces
of a
ppro
ved
enzy
mes
. Po
ssib
le a
ppli
cati
on o
f the
se g
uide
line
s to
oth
er
man
ufac
turi
ng e
nvir
onm
ents
(i.e
., fo
od p
roce
ssin
g an
d pr
otei
n ph
arm
aceu
tica
ls)
wil
l als
o be
di
scus
sed.
C-T
ER
MIN
AL
SE
QU
EN
CE
AN
ALY
SIS
OF
PO
LYP
EP
TID
ES
CO
NTA
ININ
G C
- TE
RM
INA
L P
RO
LIN
E.
Jero
me
M.
Bai
ley,
Oan
h Tu
, G
ilber
t Is
sai,
Alic
e H
a, a
nd J
ohn
E. S
hive
ly
Bec
kman
Res
earc
h In
stitu
te o
f the
City
of H
ope,
Div
isio
n of
Imm
unol
ogy,
145
0 E.
D
uart
e R
oad,
Dua
rte,
CA
91
010.
The
inab
ility
to
deri
vatiz
e C
-ter
min
al p
rolin
e ha
s be
en a
maj
or im
pedi
men
t to
the
de
velo
pmen
t of
a r
outin
e ch
emic
al m
etho
d fo
r C
-ter
min
al s
eque
nce
anal
ysis
. Pr
evio
us w
ork
in o
ur la
bora
tory
des
crib
ed c
hem
istr
y w
hich
ded
vatiz
ed t
he C
-ter
min
al
amin
o ac
id to
a t
hioh
ydan
toin
and
per
mitt
ed t
he s
eque
nce
anal
ysis
of a
ll of
the
com
mon
am
ino
acid
s w
ith t
he e
xcep
tion
of p
rolin
e. T
his
new
che
mis
try
com
bine
d th
e ac
tivat
ion
and
dedv
atiz
atio
n st
eps,
obv
iatin
g th
e ne
ed fo
r in
term
edia
te o
xazo
linon
e fo
rmat
ion.
Th
is c
hem
istr
y ha
s no
w p
erm
itted
the
dedv
atiz
aUon
and
hyd
roly
sis
of C
- te
rmin
al p
rolin
e as
a t
hioh
ydan
toin
am
ino
acid
. W
e ha
ve a
utom
ated
this
che
mis
try
with
pep
tidas
cov
alen
tly a
ttac
hed
to c
arbo
xylic
aci
d m
odifi
ed p
olye
thyl
ene
and
prot
eins
non
-cov
alen
tly a
pplie
d to
Zite
x (p
orou
s Te
flon)
. It
is n
ow p
ossi
ble,
for
the
first
tim
e, t
o ro
utin
ely
sequ
ence
thro
ugh
all t
wen
ty o
f the
com
mon
am
ino
acid
s.
Prog
ram
s an
d m
etho
ds fo
r au
tom
ated
seq
uenc
ing
are
curr
ently
bei
ng o
ptim
ized
and
will
be
pres
ente
d.
This
wor
k w
as s
uppo
rted
by
NIH
Gra
nt G
M 4
6022
.
B1
EXPLOITING AUTOMATED PROTEIN SEQUENCE ANALYSIS: IN SITU CLEAVAGE AND RE-
SEQUENCING
Alexander W. Bell, Nicole C.
Baur, John J.M. Bergeron~, Wei-Jia Ou z, David ~.
Thomas 2, Katherine Cianflone 3, Allain B
aldo 3, Maxwell T. Hincke 4, Richard
.
Momparler 5, JesSe Lalibert6 5, David M.P. Thomson% and M. Sutherland
Protein Analytic Facility,
Sheldon Biotechnelogy Centre;
IDepartment of
Anatomy; 3Division of Cardiology, Royal Victoria Hospital; & 6Department of
Immunology,
Montreal
General
Hospital;
McGill
University,
Montreal.
ZBiotechnology Research
Institute,
NRC Canada;
&
5Centre
de Recherche
Pediatrique,
H6pital
Ste-Justine,
Universitd
de
Montrdal,
Montreal.
4Department of Anatomy, University of Ottawa Faculty of Health Science,
Ottawa.
A technique i
nvolving re-sequence analysis of a sample after exposure to
conditions which a
ffect cleavage at the C-terminal side of methionyl r
esidues
is r
outinely employed a) to d
irectly assess t
he quantity eriginally s
ubjected
to sequence analysis; b) to estimate the number of cleavable methionyl
residues; c) to provide a
dditional s
equence information and/or d) to c
larify
ambiguous sequencing results. The technique involves removal of the sample
from the gas-phase sequencer followed by I) exposure to cyanogen bromide
(CNBr) in the p
resence of 70% formic a
cid a
nd 2) re-sequence analysis of the
cleavage mixture. The technique takes advantage of CNBr cleavage s
pecificity,
differential wash-out characteristics of peptldes, sequencer reliability a
nd
reproducibility, ease of manipulation and reduced labour costs related to
both sample regeneration and/or ensuing work-up. The only requirement for
success, that is the generation of new sequence information, is limited to
the s
ample c
ontaining a c
leavable methionyl residue 8-10 r
esidues from t
he C-
terminus of the protein, although the null result is informative. Several
unknown proteins, N-terminally blocked or displaying ragged N-termini have
been identified u
sing this technique.
> >
A2
B
2
STUDY
OF
A REARRANG~MENT
AT
THE
C-TERMINUS
OF
PEPTIDES
DURING
THE
COLLISION ACTIVATED DISSOCIATION EXPERIMENTS.
Vladimir
BCeada I ,
Javier
Gonzalez I ,
Gabriel
Padron I ,
Hilda
Garay I ,
Osvaldo Reyes I,
Toshifumi Takao 2,
Yasutsugu Shimonishi 2 .
i)
Center
for
Genetic
Engineering
& Biotechnology,
P.O.Box
6162,
La
Habana,
CUBA
2) Institute for Protein Research,
Osaka University,
Suita
Campus, Osaka 565, JAPAN.
An
internal
rearrangement
involving
the
loss
of
the
c-terminal
amino
acid residue of peptides in Low Energy Collision Activated Dissociation
(CAD
) experiments,
has
been
reported
i.
Specific
sequences
at
the
C-
terminus
of
peptidee
often
tend
to
be
misassigned
when
this
rearrangement
appears
as a
very intense signal
in the CAD spectrum.
We
designed
and
synthesized
a set
of
peptides
in
order
to
study
the
influence of the nature of amino acids at the rearrangement
site and the
position
of
a basic
residue
within
the
peptide
sequence
in
the
occurrence
of
this
rearrangement.
Our
results
confirm
that
this
phenomenon
is strongly
influenced
by the basicity
of
the
amino
acids
within the sequence,
thus
it could be useful
for
differentiating
two
isobaric amino acids such as Lysine and Glutamine in peptide sequencing.
Here,
we also
report
the use of
the enzymatic
18C-labeling
method
to
identify
the
C-terminal
rearrangement
of
peptides
and
avoid
misassignments of the sequence 2
l-Gaekell.
S.J et al., J.~un. Soc Mass Spectrom,
i, 249
(1990).
2-Takao. T
et al., A.Chemistry 65, 2394
(1993).
CHARACTERIZATION OF A
RECOMBINANT CYTOSOLIC DOMAIN OF CD45 PHOSPHATASE
Patricia G. Brake, Anne Pacitti, Terry Higgins, Panos Stevis~ John
Hallnowski, Sue McElhlney I,
James Huan~, Christine Vestal 3
Sterling winthrop Inc., Collegeville, PA, ZLexln Pharmaceutical Corporation,
Horsham, PA, 3PerSeptive Biosystems, Houston, TX
CD45 is a
large transmembrane glycoprotein required for antigen driven T-
and B-cell activation. The intracellular portion of CD45 contains two
protein domains one of which is an active protein tyrosine phosphatase.
The
phosphatase activity of the intraoellular portion of CD45 is believed to
initiate the cascade of events leadlng to B- and T-cell proliferation by
activating one or more src family klnases. The 82kDa cytosolic domain of
this protein was expressed at high levels in yeast and purified for use in
the initiation of biophysical studies.
But, despite an apparent purity of
this preparation on SDS-PAGE of >95%, the protein did not crystallize.
Microcharacterization of a
purified preparation of this protein by amino
acid sequence analysis and laser desorption mass spectrometry showed
heterogeneity at the N-terminus of the protein caused by cleavage at dibasic
residues.
This cleavage correlated with decreased specific activity of the
enzyme.
Subsequently, the DNA was modified by deletion of sequence coding
for residues N-terminal to the identified cleavage site and the resulting
derivative protein was expressed and purified.
Amino acid sequence analysis
showed that the identified N-terminal heterogeneity was eliminated by this
modification.
Crystallization trials of this preparation are in progress.
In addition,
SDS-PAGE analysis suggests a
small amount of internal protein
cleavage, potentially from a
similar dibasic amino acid sequence which is
present in the protein,
DNA sequence coding for this protein has been
mutated to eliminate this potential cleavage; analysis of this construction
is in progress.
t~q
B3
B5
ANAL
YSIS
OF
THE
MIC
ROHE
TERO
GEN
ITY
OF
RECO
MBI
NANT
SCH
ISTO
SOM
A MAN
SONI
ANTI
GEN
rS
mp2
8 RE
VEAL
S N-
FORM
YL-A
LANI
NE A
S A
NEW
PO
ST-T
RANS
LATI
ONA
L M
ODI
FICA
TIO
N IN
SA
CCHA
ROM
YCES
CERE
VISI
AE
Rain
er B
isch
off, D
omin
ique
Roec
klin
, Ber
nade
tte B
ouch
on, K
laus
Kla
rsko
v ~ an
d Al
ain V
an D
orss
elae
r 2
TRAN
SGEN
E S.
A., P
rote
in A
naly
tical
Gro
up, 1
1 Ru
e de
Mol
shei
m, F
-670
82 S
trasb
ourg
Ced
ex, F
RANC
E,
Vakg
roep
Bio
chem
ie Fy
siol
ogie
en M
icro
biol
ogie
, Led
egan
ckst
raat
35, 9
000 G
ent, B
ELG
IUM
, 2 Un
iver
sit(~
Loui
s Pa
steu
r, La
bora
t. de
Spe
ctro
met
rie de
Mas
se B
ioor
gani
que,
URA
31,
CNR
S, 5
Rue
Bla
ise P
asca
l, 670
08
Stra
sbou
rg C
edex
, FRA
NCE.
rSm
p28
was
exp
ress
ed in
trace
llula
rly in
a r
ecom
bina
nt s
train
of
Sacc
haro
myc
es ce
revi
siae
and
purif
ied b
y affi
nity
chr
omat
ogra
phy o
n gl
utat
hion
e-Se
phar
ose.
The
isol
ated
pro
tein
was
hom
ogen
ous
by S
DS-P
AGE
unde
r red
ucin
g co
nditi
ons
but s
how
ed h
eter
ogen
eity
upo
n is
oele
ctric
focu
sing
and
an
ion-
exch
ange
HPL
C. T
he t
wo
maj
or fo
rms
(rSm
p28/
1 an
d rS
mp2
8/2)
wer
e is
olat
ed b
y an
ion-
ex
chan
ge H
PLC
and
subi
ecte
d to
pro
tein
mic
rose
q-en
cing
reve
alin
g tha
t rSm
p28/
2 w
as N
.term
inat
ly
bloc
ked,
Com
para
tive
trypt
ic m
aps o
f the
two
form
s wer
e id
entic
al ex
cept
for o
ne a
dditi
onal
frag
men
t in
the
dige
st o
f rSm
p28/
2 w
hich
was
sho
wn
to b
e N-
term
inal
ly b
lock
ed a
nd to
cor
resp
ond
to th
e N-
te
rmin
al tr
yptic
fra
gmen
t of
rSm
p28
as d
eter
min
ed b
y am
ino
acid
ana
lysi
s, M
ass
mea
sure
men
t re
veal
ed a
diff
eren
ce o
f app
r, 26
Da
betw
een
the
expe
cted
mas
s of
the
N.te
rmin
al fr
agm
ent a
nd th
e m
easu
red
mas
s in
dica
ting
that
the
N-te
rmin
us w
as fo
rmyt
ated
(~m
= 2
8 O
a), P
artia
t deb
tock
ing
ol th
e pe
ptid
e fra
gmen
t w
ith t
riflu
oroa
cetic
aci
d al
low
ed t
o ob
tain
the
exp
ecte
d N-
term
inal
seq
uenc
e co
nfirm
ing
that
the
N-te
rmin
al a
lani
ne w
as b
lock
ed w
ith a
n ac
id la
bile
form
yl g
roup
dur
ing
prot
ein
bios
ynth
esis
in S
. cer
evis
iae,
N-fo
rmyl
-ala
nine
repr
esen
ts a
new
pos
t-tra
nsla
tiona
l mod
ifica
tion
in S
. ce
revi
siae
pote
ntia
lly im
plic
atin
g so
far n
ot d
escr
ibed
enz
ymat
ic a
ctiv
ities
.
B4
PR
EP
AR
ATI
ON
AN
D C
HA
RA
CTE
RIZ
ATI
ON
OF
THE
SIT
E-D
IRE
CTE
D E
211Q
MU
TA
NT
OF
YE
AS
T
EN
OLA
SE
1
John
M.
Bre
wer
1, V
ani
S,
San
gada
la 1
, Rob
ert
t,. R
obso
n =,
Cla
ibor
ne C
,V.
Glo
ver 1
, Mic
hael
J.
Hol
land
~,
and
Luke
sz L
ebio
da 4
~Dep
t. o
f B
ioch
emis
try,
Uni
v. G
eorg
ia,
Ath
ens,
GA
30
602;
aD
ept,
of M
icro
biol
ogy,
Uni
v. R
eadi
ng,
Eng
land
U
K;
3Dep
t. of
Bio
logi
cal
Che
mis
try,
Uni
v, C
alifo
rnia
Med
ical
Sch
ool,
Dav
is,
CA
95
616;
4D
ept.
of
Che
mis
try,
Uni
v. S
outh
Car
olin
a, C
olum
bia,
SC
29
208
Yea
st e
nola
se 1
, a
dim
eric
enz
yme,
bin
ds o
ne M
g++
/sub
unit
enab
ling
subs
trat
e bi
ndin
g, w
hich
in
turn
ena
bles
a s
econ
d M
~3~ *
lsub
unit
bin
ding
and
cat
alys
is I
lh
The
'ch
arge
shu
ttle
' m
echa
nism
of
enol
ase
cata
lysi
s, b
ased
on
X-r
ay c
ryst
allo
grap
hic
stud
ies,
inv
olve
s tr
ansf
er o
f th
e pr
oton
on
carb
on-2
o
f sa
bstr
ete
to a
H2O
, the
n to
the
car
boxy
late
of
Glu
-168
, ov
er t
o th
e ca
rbox
ylat
e o
f G
lu-2
11 a
nd
subs
eque
ntly
to
the
solv
ent
(2).
T
o te
st t
he i
nvol
vem
ent
of G
lu-2
11 i
n th
is m
echa
nism
a s
ite-d
irec
ted
mut
ant,
E21
1Q,
wa
s pr
epar
ed a
nd c
hara
cter
ized
as
desc
ribe
d fo
r E
168Q
(3)
. T
he i
dent
ity o
f E
211Q
w
as c
onfir
med
by
SD
S-g
el e
lect
roph
ores
is a
nd a
min
o ac
id s
eque
ncin
g.
E21
1Q s
how
ed 0
.O1%
of
the
na~L
ve e
nzym
e ac
tivity
in t
he s
tand
ard
assa
y,
Diff
eren
tial s
cann
ing
calo
rim
etry
(D
SC
) sh
owed
tha
t E
211Q
and
nat
ive
enol
ases
res
pond
sim
ilarl
y to
Mg
++ a
nd s
ubst
rate
. A
ll th
e E
211Q
enz
yme
is f
olde
d co
rrec
tly.
Spe
ctro
phot
omet
ric
titra
tions
wit
h a
sub
stra
ta a
nalo
gue,
D-t
artr
onat
e se
mia
ldeh
yde-
2-
phos
phat
e (4
) co
nfir
med
tha
t th
e bi
ndin
g af
fini
ty w
as n
ot a
ffec
ted
in t
he m
utan
t.
Rea
ctio
n o
f th
e an
alog
ue w
ith
E21
IQ
sug
gest
s pa
rt o
f th
e re
actio
n is
abo
ut t
wo
ord
ers
of
mag
nitu
de s
low
er
than
th
at w
ith
the
nat
ive
enzy
me,
T
hese
obs
erva
tions
are
con
sist
ent
wit
h t
he p
redi
cted
par
ticip
atio
n o
f G
lu-2
11 i
n th
e 'c
harg
e sh
uttle
' m
echa
nism
. 1.
B
rew
er,
J.M
. C
rit.
Rev
. B
ioch
emis
try
11
209
(18
81).
2.
Le
biod
a, L
. an
d S
tec,
B.
Bio
chem
istr
y 30
281
7 (1
991)
. 3.
B
rew
er,
J.M
., R
obso
n, R
.L.,
Glo
ver,
C.V
.C.,
Hol
land
, M
.J.
and
Lebi
oda,
L.
P
rote
ins:
S
truc
ture
, F
unct
ion
and
Gen
etic
s 1
7 4
26 (
1993
).
4.
Spr
ing,
T.G
. an
d W
old,
F,
Bio
chem
istr
y 1
0 4
655
(197
1).
86
Dir
ect
Mas
s Sp
ectr
omet
ric
Ana
lyse
s fo
r P
rote
in C
hem
istr
y St
udie
s
Sco
tt D
. B
ucke
l, T
racy
Ste
vens
on a
nd J
osep
h A
. L
oo,
Par
ke-D
avis
Pha
rmac
euti
cal
Res
earc
h,
Div
isio
n of
War
ner
Lam
bert
Com
pany
, A
nn A
rbor
, M
I 48
105
In o
ur s
tudi
es o
f th
e st
ruct
ure
of v
ario
us p
rote
ins,
we
have
use
d a
com
bin
atio
n o
f pr
otei
n se
quen
ce a
naly
sis,
mat
rix-
assi
sted
las
er d
esor
ptio
n/io
niza
tion
(M
AL
DI)
ti
me-
of-f
ligh
t m
ass
spec
trom
etry
and
ele
ctro
spra
y io
niza
tion
(E
SI)
mas
s sp
ectr
omet
ry.
MA
LD
I-M
S i
s le
ss s
ensi
tive
to
the
pres
ence
of
extr
aneo
us s
alts
tha
n E
SI.
H
owev
er,
the
abil
ity
to d
irec
tly
mas
s an
alyz
e pr
otei
n m
ater
ials
via
ES
I w
itho
ut a
ddit
iona
l sa
mpl
e cl
eanu
p pr
oced
ures
res
ults
fro
m t
he d
iscr
imin
atin
g na
ture
of
a fo
cal-
plan
e m
ay
dete
ctor
. T
he a
rray
det
ecto
r ca
n be
"tu
ned"
for
lo
w l
evel
, hi
gher
ch
arge
d sp
ecie
s in
a c
ompl
ex m
ixtu
re a
nd]o
r in
the
pre
senc
e of
low
mol
ecul
ar m
ater
ial.
M
AL
DI-
MS
ca
n be
u
sed
to
m
on
ito
r th
e d
egre
e o
f tr
un
cati
on
an
d
the
seq
uen
ce
by
carb
oxyp
epti
dase
tre
atm
ent
of p
epti
des
and
smal
l pr
otei
ns w
itho
ut r
emo
val
of
extr
aneo
us b
uffe
r m
ater
ials
. T
he
cap
abil
ity
of
anal
yzi
ng
pro
tein
s se
para
ted
by
elec
tro
ph
ore
sis
wit
h
mas
s sp
ectr
omet
ry i
s a
pow
erfu
l to
ol.
We
have
bee
n ab
le t
o de
term
ine
the
mo
lecu
lar
wei
ghts
of
spot
s fr
om t
wo-
dim
ensi
onal
gel
s by
ES
I th
at h
ad b
een
blot
ted
onto
PV
DF
mem
bran
es,
stai
ned
wit
h C
oo
mas
sie
blue
, an
d ex
trac
ted
wit
h he
xafl
uoro
isop
ropa
nol
(HF
IP),
an
d a
lso
det
erm
ined
the
se
quen
ce o
f pe
ptid
es v
ia c
hem
ical
dig
esti
on o
f th
e m
ater
ial
not
used
for
mas
s de
term
inat
ion.
W
e ha
ve t
aken
sam
ples
dir
ectl
y fr
om a
n af
fini
ty c
olum
n el
uate
und
er h
igh
salt
con
diti
ons,
bou
nd t
he
prot
ein
to t
he m
emb
ran
e in
a P
roS
pin|
ca
rtri
dge,
ext
ract
ed t
he m
emb
ran
e w
ith
HF
IP,
and
dete
rmin
ed t
he m
ass
of
prot
ein
by M
AL
DI-
MS
and
ES
I-M
S.
The
nu
mb
er o
f di
sulf
ide
bond
s pr
esen
t in
sm
all
tig
hd
y-b
rid
ged
pep
tide
s ca
n be
det
erm
ined
by
the
dif
fere
nce
in m
ass
of t
he
oxid
ized
mat
eria
l an
d th
e m
ater
ial
redu
ced
by t
ris-
(2-c
arb0
xyet
hyl)
phos
phin
e w
itho
ut a
ddit
iona
l de
riva
tiza
fion
of
the
resu
ltin
g fr
ee c
yste
ines
..
B7
8~IA
ME
MB
RA
NE
-AN
D M
ICR
OFI
LA
ME
NT
-ASS
OC
IAT
ED
gag
-LIK
E P
RO
TE
IN F
RO
M A
SC1T
ES
OR
CE
LL
MIC
RO
VIL
LI,
BIN
DS
SR
C S
H3
DO
MA
IN T
HR
OU
GH
A P
OL
Y-P
RO
LIN
E M
OT
IF
C.A
. C
arot
hers
Car
raw
ay, l
J. H
uang
, l Y
. Li,
t S.-
H.
Juan
g, 1
A.
Gal
hi, 1
B.J
. May
er, 3
andK
. L
. Car
raw
ay. 2
D
epts
. of
Bio
chem
. &
Mol
ec.
Bio
l) a
nd.C
eU B
iol.
& A
nat.
,2 U
niv.
of
Mia
mi S
ch.
of M
edic
ine,
Mia
mi,
F
L 3
3136
and
The
Roc
kefe
ller
Inst
itute
," N
ew Y
ork,
NY
100
21
Asc
ites
subl
ines
of t
he 1
3762
rat
mam
mm
'y ad
enoc
arci
nom
avro
vide
a us
eful
mod
el s
yste
m fo
r inv
esti
gati
ng
mem
bran
e-m
iero
fila
men
t (M
F) i
nter
acti
ons t
. T
he M
AT
-Cf
subl
ine
diff
ers
from
til
e M
AT
-BI
subf
fam
in
havi
ng h
ighl
y st
able
, bra
n~he
r m
icro
vill
i (M
V) 2
and
im
mob
ile
cell
sur
face
rec
epto
rs. 3
A 5
8 kD
a pr
otei
n (p
58),
exp
ress
ed in
the
MA
T-C
1 bu
t not
tlie
MA
T-B
1 ce
lls 4
, has
bee
n im
plic
ated
in th
e st
abil
izat
ion o
f the
~
AT
-C1
cell
sur
face
, v5
8 is
pre
sent
in
MV
in
a tr
ansm
embr
ene
com
vl~x
(TM
C) 5
wit
h ac
tin
and
a hi
gh
M,
com
plex
of
at l
east
"fiv
e gi
ycop
rote
ins
(55,
65,
80,
110
and
120
kO
a). 6
The
TM
C s
erve
s as
the
cor
e fo
r a
larg
e si
gnal
tran
sduc
tion
pa~
cle
cont
aini
ng p1
85m
/Erb
B27
and
sev
eral
kno
wn
sign
al tr
ansd
ucti
on
com
pone
nts,
[nc
hidi
ng p
60~.
=. P
urif
ied
p58
bind
s -ol
iosv
holiv
ids
and
bloc
ks a
ctin
pol
ymer
]zai
lon,
act
ing
as
a fi
lam
ent c
appi
ng p
rote
in.4
The
se m
embr
ane-
and
MI*
-bin
ding
pro
pert
ies
are
cons
iste
nt w
ith
the
prop
osed
ro
le o
f p58
in s
tabi
lizi
ng th
e hi
ghly
mal
igna
nt M
AT
-C1
cell
suTr
face
by s
tabi
lizin
g, th
e in
tera
ctio
ns b
etw
een
the
mie
rovi
llar
MF
s an
d th
e m
embr
ane.
T
he c
ompl
ete
eDN
A s
eque
nce
of p
5~ h
as b
een
obta
ined
S, e
nd
the
dedu
ced
amin
o ad
td s
eque
m~
sho
wed
a s
urpt
lsin
g ~
_tt_
~i" ty
te m
anim
alia
n re
trov
lral
sag
__
l~3~_
, i
nclu
ding
re
gion
s cor
resp
ondi
ng tu
p15
, pl2
mtd
the
N-t
erm
inal
80%
of p
30 b
ut n
o p
l0 s
eque
nce,
p58
als
o co
ntai
ned
a se
quen
ce 0
PPPY
PVPT
APP
) ~t
4thi
n a
long
er p
roli
ne-r
leh
sequ
ence
sim
ilar
to t
hose
fo~n
d in
Sro
and
AIM
SH
3 do
mai
n-bi
ndin
g pr
otei
ns 3
BPI
and
3B
P2.
p58
boun
d to
Sro
SH
3 do
mai
n on
blo
ts w
ith
biot
i93~
late
d SH
3 do
mai
n an
d on
SH
3 do
mai
n-ag
aros
e of
a m
ic~v
illa
r ext
ract
. In
viw
o-tr
ansl
ated
p58
eo-
imm
unop
reci
#ita
ted
wit
h pl
atel
et c
-Src
in
the
abse
nce
but n
ot i
n th
e pr
esen
ce o
f sy
nOet
ic p
epil
de.
The
se r
esul
ts s
ugge
st th
at
p58
is a
n im
port
ant e
lem
ent i
n or
gani
zing
a M
F-as
soci
ated
si~
ml t
rans
duct
ion
part
icle
at t
he c
ytop
lasm
ic
surf
ace
of th
e pl
asm
a m
embr
ane
hi th
e as
clte
s cel
ls.
We
prop
ose
and
are
test
ing
ihe
hypo
thes
is th
at b
indi
ng
of p
58 t
o Sr
c m
ay a
lso
mod
ulat
e Sr
c ac
tivi
ty a
nd s
igns
[ trd
nsdu
ctio
n in
the
tum
or 6
ells
.
1.
C. C
arra
way
and
K.
Car
raw
ay (1
989)
Bio
chim
. Bio
phys
. Act
a B
iom
embr
anes
Rev
iew
s 988
,147
-171
. 2.
K
. C
arra
way
et
al.
(198
0)
Nat
ure
285,
508
-510
. 3.
K
. C
arra
way
et
al.
(197
9)
J. C
ell B
iol.
83,5
29-5
43.
4.
Y.
Liu
et
aI.
(198
9)
J. B
iol.
Che
m.
264,
120
8-1,
214.
5.
C
. C
arra
way
et
al.
(198
3)
Proc
. N
ail.
Aca
d. S
ci.
80,4
30-4
34).
6.
C
. C
arra
way
et
al.
(199
1)
J. B
iol.
Che
m.
266,
162
38-1
6246
. 7.
C
. C
arra
way
et
al.
(199
3)
J. B
iol.
Che
m.
268,
558
2-55
87.
8.
S.-H
. Ju
ang
at a
l.
(199
4) J
. B
iol.
Che
m.,
in p
ress
.
C2
STR
UC
TUR
E C
HAR
ACTE
RIZ
ATIO
N O
F M
EMBR
ANE B
OU
ND
AN
D S
UR
FAC
E AD
SOR
BED
PR
OTE
IN
Mar
tin C
affre
y, J
in W
ang,
Car
mic
hael
J.
A.
Wal
lace
1,
lan
Cla
rk-L
ewis
2 D
ept.
of C
hem
istry
, Th
e O
hio
Sta
te U
nive
rsity
, C
olum
bus,
OH
432
10.
U.S
.A.,
1Dep
t. of
B
ioch
emis
try,
Dal
hous
ie U
nive
rsity
, H
alifa
x, N
ova
Sco
tia,
Can
ada
B3H
4H
7, 2
Bio
med
ical
R
esea
mh
Cen
ter,
Uni
vers
ity o
f B
ritis
h C
olum
bia,
Van
couv
er,
B.C
., C
anad
a V
6T 1
W5.
The
x-ra
y st
andi
ng w
ave
(XS
W)
met
hod
(1-6
) ha
s be
en u
sed
to s
tudy
the
stru
ctur
e an
d to
polo
gy o
f th
e pr
otei
n, c
ytoc
hrom
e c,
bou
nd to
a n
egat
ivel
y ch
arge
d m
odel
lipi
d m
embr
ane
and
adso
rbed
at
a m
etal
sur
face
. A
t the
met
al s
urfa
ce,
cyto
chro
me
c fo
rms
an h
exag
onal
ly
clos
e-pa
cked
mon
olay
er.
A s
imila
r pa
ckin
g ar
rang
emen
t is
obs
erve
d at
the
sur
face
of
a se
lf-as
sem
bled
lip
id m
onol
ayer
on
silv
er.
In t
he c
ase
of a
Lan
gmui
r-B
lodg
ett
(LB
) fil
m,
prot
ein
mul
tilay
ers
are
form
ed.
The
data
sug
gest
tha
t cy
toch
rom
e c
mai
ntai
ns i
ts n
ativ
e gl
obul
ar s
truct
ure
upon
sur
face
bin
ding
an
d su
bseq
uent
sto
rage
for
an
exte
nded
per
iod.
Fu
rther
, th
e da
ta a
re c
onsi
sten
t w
ith a
pro
tein
doc
king
mec
hani
sm w
here
in th
e he
me
plan
e is
orie
nted
per
pend
icul
ar t
o an
d w
ith i
ts e
xpos
ed e
dge
faci
ng t
he s
urfa
ce.
This
stu
dy
dem
onst
rate
s th
e ut
ility
of
XS
Ws
as a
new
and
pow
erfu
l st
ruct
ural
too
l fo
r in
vest
igat
ing
mem
bran
e- a
nd s
urfa
ce-a
ssoc
iate
d pr
otei
ns.
1.
M.J
. B
edzy
k et
al..
Sci
ence
241,
17
88 (
1990
) 2.
M
.J.
Bed
zyk
et a
l., S
cien
ce 2
48,
52 (
1990
) 3.
M
. C
affre
y et
al.,
Far
aday
Dis
cuss
. 94,
In
pres
s (1
993)
4.
J.
Wan
g et
al..
Nat
ure
354,
377
(19
91)
5.
J. W
ang
et a
l.. S
cien
ce 2
58,
775
(199
2)
6.
Jin
Wan
g et
al..
J.
Mol
. B
iol.
In p
ress
.
C1
DIR
EC
T D
ET
ER
MIN
AT
ION
O
F IM
MO
BIL
IZE
D
PR
OT
EIN
CO
NC
EN
TR
AT
ION
.
Pat
rick
L.
Col
eman
& D
anie
l S
arpo
ng.
Bio
scie
nces
Lab
orat
ory.
3M
Co.
St.
Pau
l, M
N
One
of
the
mos
t fu
ndam
enta
l ne
eds
in b
ioch
emic
al r
esea
rch
is k
now
ledg
e of
the
co
ncen
trat
ion
of p
rote
in.
Thi
s is
gen
eral
ly n
ot t
oo d
iffic
ult
whe
n th
e pr
otei
n is
in
solu
tion,
whe
n an
y on
e of
sev
eral
diff
eren
t op
tical
or
chem
ical
met
hods
can
giv
e re
liabl
e re
sults
. In
con
tras
t, w
ork
with
im
mob
ilize
d pr
otei
ns i
s of
ten
ham
pere
d by
in
abili
ty to
acc
urat
ely
dete
rmin
ethe
mas
s of
pro
tein
pre
sent
. M
any
of th
e co
mm
on
supp
ort
mat
eria
ls p
recl
ude
the
use
of o
ptic
al m
etho
ds.
We
desc
ribe
in
this
pos
ter
a di
rect
che
mic
al m
etho
d fo
r th
e de
term
inat
ion
of th
e co
ncen
trat
ion
(den
sity
) of
pro
tein
cou
pled
to
seve
ral t
ypes
of
poro
us m
edia
freq
uent
ly
used
as
supp
orts
for
im
mob
iliza
tions
in a
ffin
ity s
epar
atio
ns a
nd e
nzym
ic c
atal
ysis
. T
he
met
hod
empl
oys
the
read
ily a
vaila
ble
BC
A (
bici
ncho
nini
c ac
id)
whi
ch i
nter
acts
with
th
e C
u+ f
orm
ed b
y re
duct
ion
of C
u2+
in t
he p
rese
nce
of p
rote
in a
mid
e bo
nds
(Ana
l. B
ioch
em.
150,
76,
198
5).
The
rea
ctio
n ta
kes
plac
e at
roo
m t
empe
ratu
re a
nd c
an b
e re
ad in
as
little
as
30 m
in.,
thou
gh m
ore
typi
cally
in 2
h.
The
ass
ay is
sen
sitiv
e to
as
little
as
50 I
lg o
f im
mob
ilize
d pr
otei
n an
d is
gen
eral
ly in
depe
nden
t of
the
am
ount
of
supp
ort
pres
ent.
G
reat
er s
ensi
tivity
is f
easi
ble
but
has
not
yet
prov
en n
eces
sary
. T
he
assa
y ha
s pr
oved
inv
alua
ble
in o
ptim
izin
g th
e ac
tivity
of
imm
obili
zed
prot
ein
ligan
ds
for
affin
ity s
epar
atio
ns,
sinc
e hi
gh c
oupl
ing
effic
ienc
es m
ake
use
of in
dire
ct m
etho
ds
(det
erm
inat
ion
of t
he a
mou
nt o
f pr
otei
n w
hich
has
not
cou
pled
) hi
ghly
ina
ccur
ate.
W
e de
velo
ped
the
met
hod
in c
onju
nctio
n w
ith E
mph
aze T
M B
iosu
ppor
t M
ediu
m,
but
have
de
term
ined
tha
t it
is w
idel
y ap
plic
able
to
mos
t of
the
com
mer
cial
ly a
vaila
ble
affin
ity
supp
orts
. C
3
Con
form
atio
nal
Fle
xibi
lity
of
th
e G
ly-G
ly
dipe
ptid
e w
ithi
n pr
otei
n st
ruct
ures
, ti
t
Dav
id W
. Dee
rliel
d 111
Am
anda
Hol
land
-Min
kley
1, Jo
hn D
. Hem
pel 2
and
Hug
h B
. Nic
hola
s Jr.
1
1 .) P
ittsb
urgh
Supe
rcom
putin
g Cen
ter,
4400
Fif
th A
venu
e. P
ittsb
urgh
, PA
152
13
2.) D
epar
tmen
t of M
olec
ular
Gen
etic
s/B
ioch
emis
try, U
nive
rsity
of P
ittsb
urgh
, Pitt
sbur
gh, P
A 1
5261
We e
xam
ined
the
conf
orm
atio
n of d
ipep
tides
segm
ents
from
ove
r 100
pro
tein
s con
tain
ed w
ithin
the
Prot
ein
Dat
a Ban
k as d
efin
ed by
Qia
n an
d Se
jnow
ski (
J. M
ol. B
iol.
1988
, 202
, 865
-884
). W
e in
itial
ly cr
eate
d a
Prot
ein S
truct
ure d
ata b
ase (
PSdb
) ent
ry (i
nclu
ding
mol
ecul
ar su
rfac
e acc
essi
bilit
y, se
cond
ary s
truc
ture
in
form
atio
n and
oth
er de
scrip
tors
) for
each
of t
he p
rote
ins w
ith ea
ch re
sidu
e's c
onfo
rmat
ion c
lass
ified
ac
cord
ing t
o Sc
hera
ga's d
efin
ition
s of p
eptid
e con
form
atio
nal a
ngle
s (M
acro
j~ol
ecul
es 19
83, 1
6, 1
043-
10
49).
We t
hen s
earc
h thr
ough
the P
Sdb e
ntri
es de
term
inin
g the
rela
tive f
requ
ency
for e
ach
of th
e va
riou
s co
nfor
mat
ions
for b
oth m
ono-
and d
ipep
tides
. We
obse
rved
the s
ame g
ener
al pe
ptid
e con
form
atio
na]
tend
enci
es re
porte
d by
Kam
imur
a and
Tak
ahas
hi (C
AB
IO$1
994,
10, 1
63-1
69).
We d
ivid
ed th
e R
araa
chan
dran
plot
for e
ach r
esid
ue in
to h
igh
ener
gy (g
? > 0
) and
low
ener
gy (~
< 0
) are
as w
hich
, for
the
dipe
ptid
e, yi
elds
four
dist
inct
ener
gy qu
adra
nts (
L-L
, H-L
, L-H
, H-H
). T
he "t
ypic
al" d
ipep
tide s
egm
ent (
for
non-
glyc
ine d
ipep
tides
) was
foun
d to
be: L
-L=9
1%, H
-L=4
%, L
-H=4
% an
d H
-H=I
%. F
or G
ly-G
ly, th
ese
num
bers
wer
e: L
-L=2
4%, H
-L=2
7%, L
-H=I
9%, H
-H=3
0%. T
he fr
eque
ncy f
or a
sing
le G
ly re
sidu
e was
H
=53%
and L
=47%
. Thu
s, fo
r the
Gly
-Gly
dipe
pfid
e seg
men
t in p
rote
ins,
the
stat
istic
ally
expe
cted
and
obse
rved
valu
es w
ere f
ound
to b
e sim
iliar
with
in ex
pect
ed er
ror.
Thi
s ind
icat
es th
at th
ere
is n
ot a
syne
rgis
tic
influ
ence
on m
ainc
hain
conf
orm
atio
ns of
Gly
-Gly
segm
ents
; but
rath
er, i
s sh
nply
the p
rodu
ct o
f the
co
nfor
mat
iona
l flex
ibili
ty of
each
resi
due.
D1
A
HIG
HL
Y,
CO
NS
ER
VE
D
N-T
ER
MIN
AL
S
EQ
UE
NC
E
FO
R
TE
LE
OS
T
VIT
EL
LO
GE
NIN
S
Nan
cy D
. D
ensl
ow,
Ler
oy C
. F
olm
ar t,
Cra
ig V
. S
ulli
van 2
, D
ept.
Bio
ehem
. an
d M
ol.
Bio
l.,
Uni
vers
ity
of F
lori
da,
Gai
nesv
ille
, F
L,
tU.S
. E
nvir
onm
enta
l Pro
tect
ion
Age
ncy,
G
ulf
Bre
eze,
FL
, an
d 2D
ept.
Zoo
logy
, N
orth
Car
olin
a St
ate
Uni
vers
ity,
Ral
eigh
, N
C.
N-t
erm
inal
am
ino
acid
seq
uenc
es w
ere
obta
ined
for
vit
ello
geni
n (V
tg)
from
sev
eral
ph
ylog
enet
ieal
ly d
iver
se fi
sh in
clud
ing:
str
iped
bas
s (M
oron
e sax
atili
s), p
infi
sh (L
agod
on
rhom
boid
es),
brow
n bu
llhe
ad (
Amei
urus
neb
ulos
us),
med
aka
(Ory
zias
lat
ipes
), an
d ye
llow
per
ch (
perc
afla
vesc
ens)
. Th
ese
sequ
ence
s ar
e co
mpa
re~
wit
h se
quen
ces
deri
ved
from
eD
NA
of
m
umm
icho
g (F
undu
lus
hete
rocl
itus)
and
st
urge
on
(Aci
pens
er
tran
smon
tanu
s) a
nd w
ith
publ
ishe
d N
-ter
min
al a
min
o ac
id s
eque
nces
fro
m t
he l
ampr
ey
(Ict
hyom
yzon
uni
cusp
is),
claw
ed f
rog
(Xen
opus
lae
vis)
and
dom
esti
c ch
icke
n (G
allu
s do
mes
tica)
. A
seg
men
t bet
wee
n am
ino
acid
s 7
and
20 i
s hi
ghly
sii
nila
r am
ong
the
fish
sp
ecie
s.
Usi
ng
the
stri
ped
bass
se
quen
ce
as
the
tem
plat
e fo
r co
mpa
riso
ns,
the
mum
mic
hog
show
ed 1
00%
ide
ntit
y in
thi
s se
gmen
t,
pinf
ish,
87%
; br
own
bull
head
s,
93%
, w
hite
stu
rgeo
n, 6
0% a
nd s
ilve
r la
mpr
ey 4
7%.
The
yel
low
per
ch a
nd m
edak
a sh
owed
10
0%
iden
tity
wit
h th
e st
ripe
d ba
ss b
etw
een
posi
tion
s 5
and
10.
We
have
m
odel
ed a
pep
tide
to
the
cons
erve
d re
gion
s in
thi
s se
quen
ce a
nd h
ave
used
it t
o m
ake
a po
lycl
onal
ant
ibod
y in
rab
bits
. T
he a
ntib
ody
has
a w
ide
cros
s-re
acti
vity
to v
itel
loge
nin
from
dif
fere
nt s
peci
es a
nd m
ay b
e a
usef
ul d
iagn
osti
c to
ol t
o ex
amin
e vi
tell
ogen
esis
in
fish
.
D2
KA
PID
~FIN
GE
RP
RIN
TIN
G O
F P
RO
TE
INS
WIT
H I
MM
OB
IL~
D
PR
OT
EA
SE
CO
LU
MN
S
Jam
es D
. D
ixon
, Jo
nath
an P
. M
ark,
Chr
isto
pher
P. E
lico
ne,
Sim
in D
. M
alek
nia,
Bri
an F
. M
cGui
nnes
s, F
red
E.
Reg
nier
and
Nou
bar
B.
Afe
yan
Per
Sep
tive
Bio
syst
ems,
Inc.
38
Sid
ney
Stre
et,
Cam
brid
ge, M
A 0
2139
We
desc
ribe
an
appr
oach
uti
lizi
ng ir
mno
bilD
ed p
rote
ase'
s fo
r ra
pidl
y ge
nera
ting
repr
oduc
ible
pr
oteo
lyti
c di
gest
s of
pro
tein
s. T
hese
pro
teol
ytic
dig
ests
are
then
ana
lyze
d us
ing
high
per
form
ance
li
quid
chr
omat
ogra
phy
(HP
LC
) or
mas
s sp
ectr
omet
ric
(MS
) te
chni
ques
to f
inge
rpri
nt th
e pr
otei
ns.
The
pro
cedu
re h
as b
een
auto
mat
ed u
sing
a m
ulti
-col
umn
swit
chin
g de
vice
to s
elec
tive
ly p
lace
in-
line
a v
arie
ty o
fim
mob
iliT
ed p
rote
ase
colu
mns
. Im
mob
iliz
ed p
rote
ase
colu
mns
con
tain
ing
tryp
sin,
ct
-chy
mot
ryps
in, c
ucum
isin
, pap
ain,
pep
sin,
and
end
opro
tein
ase
Lys
-C h
ave
been
eva
luat
ed i
n th
is
conf
igur
atio
n. T
he p
rote
olyt
ic d
iges
tion
pro
duct
s ar
e di
rect
ly c
aptu
red
on a
C-1
8 re
vers
e-ph
ase
colu
mn
for
a su
bseq
uent
HP
LC
pep
tide
map
ping
ste
p.
The
imm
obil
ized
pre
tens
e co
lum
ns a
re
stab
le to
cha
otro
pic
solu
tion
s su
ch a
s 3.
0 M
gua
nidi
ne h
ydro
chlo
ride
or
4.0
M u
rea.
At
a te
mpe
ratu
re o
f 60
oc,
com
plet
e di
gest
s o
f pr
otei
ns a
re g
ener
ated
in
unde
r on
e m
inut
e w
ith
the
imm
obil
ized
pre
tens
e co
!um
ns.
Fur
ther
mor
e, p
assi
ve s
ampl
e lo
sses
due
to a
dsor
ptio
n to
su
rfac
es d
urin
g m
anua
l sam
ple
man
ipul
atio
ns a
re r
educ
ed b
y us
ing
the
tand
em c
olum
n ca
ptur
e ap
proa
ch.
Ove
r on
e th
ousa
nd s
ampl
e in
ject
ions
hav
e be
en l
ogge
d on
a s
ingl
e co
lum
n ov
er a
pe
riod
of
seve
ral w
eeks
.
Rap
id,
repr
oduc
ible
pro
teol
ytic
dig
ests
of
prot
eins
are
obt
aine
d us
ing
thes
e im
mob
iliz
ed p
rote
ase
colu
mns
. T
he d
iges
ts a
re s
uita
ble
for
use
in c
ompa
rati
ve p
epti
de m
appi
ng a
ppli
cati
ons
or f
or M
S
anal
yses
to g
ener
ate
mas
s m
aps
for
data
base
sea
rchi
ng.
D3
PR
OTE
IN: F
AR
NE
SY
L TR
AN
SFE
RA
SE
. E
VID
EN
CE
FO
R A
N E
LEC
TRO
PH
ILIC
S
UB
STI
TUTI
ON
ME
CH
AN
ISM
.
Julia
M.
Dol
ence
and
C.
Dal
e P
oulte
r. D
epar
tmen
t o~
Che
mis
try, U
nive
rsity
of U
tah.
Pro
tein
pre
nyl t
rans
fera
ses
cata
lyze
the
post
tran
slat
iona
l m
odifi
catio
n of
a w
ide
varie
ty o
f pro
tein
s, in
clud
ing
fung
al m
atin
g fa
ctor
s, n
ucle
ar la
min
s, a
nd R
as.
Pro
tein
: fa
rnes
yl tr
ansf
eras
e (P
FTas
e) c
atal
yzes
the
trans
fer
of a
farn
esyl
gro
up to
a c
yste
ine
resi
due
form
ing
a th
ioet
her b
ond.
A
ser
ies
of fa
rnes
yl d
ipho
spha
te (F
PP
) an
alog
s co
ntai
ning
fluo
rom
ethy
l, tri
fluor
omet
hyl,
and
hydr
ogen
at t
he 3
' pos
ition
wer
e sy
nthe
size
d an
d us
ed a
s al
tern
ate
subs
trate
s an
d in
hibJ
tors
. M
axim
um v
eloc
ities
and
M
icha
elis
con
stan
ts w
ere
mea
sure
d, a
nd th
e re
sults
sup
port
an e
lect
roph
ilic
alky
latio
n m
echa
nism
, si
mila
r to
oth
er p
reny
l enz
ymes
invo
lvin
g al
kyla
tions
of c
arbo
n-ca
rbon
do
uble
bon
ds.
Res
ults
from
thi
s st
udy
may
be
exte
nded
to u
nder
stan
ding
the
m
echa
nism
of o
ther
pre
nyl t
rans
fera
se e
nzym
es.
D4
DIS
UL
PH
ID. E
M
AP
PIN
G
OF
PR
OL
AM
INS
Ts
ez
i E
g~
rov
, A
lex
an
de
r M
us
~ly
am
ov
, Y
ve
s
Po
pin
ea
u I
,
Je
ns
A
nd
ers
en
,
Pe
ter
Ro
ep
sto
rff
An
aly
tic
al
Pro
tein
C
he
mis
try
R
es
ea
rch
G
rou
p,
En
ge
l~a
rdt
Ins
t.
of
Mo
lec
ula
r B
iolo
gy
R
us
sia
n
Ac
ad
o
f S
ci.
, R
us
sia
, 2
-1N
RA
, L
ab
. o
f B
ioc
he
mis
try
a
nd
T
ec
hn
olo
gy
o
f P
rote
ins
, F
ran
ce
, P
rote
in
Re
se
arc
h
Gro
up
, D
ep
t.
of
Mo
lec
ula
r B
iolo
gy
, O
de
ns
e
Un
iv.,
D
en
ma
rk
Pro
lam
ins
, th
e
ma
jor
ce
rea
l s
ee
d
sto
rag
e
pro
tein
s,
co
mp
ris
e
a
fam
ily
of
h
ete
rog
en
eo
us
p
rote
ins
w
ith
co
mm
on
p
rop
ert
ies
, a
mo
un
d
wh
ich
s
olu
bil
ity
in
a
q.
alc
oh
ol
an
d
hig
h
glu
tam
in
an
d
pro
lin
c
on
ten
ts.
Th
e
pu
rpo
se
o
f th
is
stu
dy
Is
th
e
ide
nti
fic
ati
on
o
f in
tra
- a
nd
in
ter-
c
ha
in
dis
ulp
hid
e
bo
nd
s
of
wh
ea
t p
rola
min
s
mo
no
me
ric
g
lla
din
s
, a
s
we
ll
as
of
oa
t o
ne
s
(av
en
ins
).
Th
e
co
va
len
t c
hro
ma
tog
rap
hy
,
HP
LC
, g
el
ele
ctr
op
ho
res
is,
mlc
ros
eq
ue
n-
cin
g
an
d
ma
ss
sp
ec
tro
me
try
w
ere
u
se
d
in
this
s
tud
y
for
pro
tein
p
uri
fic
ati
on
a
nd
d
isu
lph
ide
m
ap
pin
g
of
pro
lam
ins
. A
s
a
res
ult
, s
ev
era
l p
ure
p
rola
min
c
om
po
ne
nts
h
av
e
be
en
is
ola
ted
a
nd
c
ha
rac
teri
ze
d
(1).
T
he
e
xa
ct
nu
mb
er
of
cyst
ein
e re
sid
ue
s
In
pro
lam
lns
w
as
de
term
ine
d
by
ES
MS
b
efo
re
an
d
aft
er
red
uc
tio
n
an
d
alk
yla
tio
n.
All
S
-S-b
on
ds
o
f a
ve
nin
-3
(2)
an
d
~-4
B
gli
ad
in
we
re
ide
nti
fie
d.
I.
T.
Eg
oro
v
et
al~
, J.
Ce
rea
l.
Sc
i.
(ac
ce
pte
d).
2.
T.
E
go
rov
e
t a
l.,
Eu
r.
J.
Bio
ch
em
, (a
cc
ep
ted
).
MA
PP
ING
AN
D C
HA
RA
CT
ER
IZA
TIO
N O
F P
RO
TE
INS
AS
SO
CIA
TE
D W
ITH
M
OR
PH
OL
OG
ICA
L T
RA
NS
FO
RM
AT
ION
IN
TH
E S
HE
CE
LL
AS
SA
Y.
D. L
. G
augg
el, T
. N
. Asq
uith
, R. J
. Isf
ort,
N.
S. M
ille
r an
d D
. B.
Cod
y C
orpo
rate
Pro
fess
iona
l & R
egul
ator
y Se
rvic
es/H
uman
Saf
ety
Dep
t., T
he P
roct
er &
G
ambl
e C
o., P
.O.
Box
398
707,
Cin
cinn
ati,
Ohi
o, 4
5239
-870
7.
The
Syr
ian
Ham
ster
Em
bryo
(SH
E)
Cel
l Ass
ay is
a g
ood
scre
en fo
r pot
enti
al
carc
inog
enic
act
ivit
y of
che
mic
als.
Und
er re
duce
d pH
cel
l cul
ture
con
diti
ons,
it d
etec
ts
both
gen
otox
ic a
nd n
onge
noto
xic
carc
inog
ens
wit
h an
ove
rall
con
cord
ance
of
86%
whe
n co
mpa
red
to c
hem
ical
s te
sted
in a
nim
al m
odel
s. C
hang
es in
cel
lula
r mor
phol
ogy
are
used
to a
sses
s ca
rcin
ogen
ic a
ctiv
ity.
Pro
tein
s fr
om n
orm
al a
nd tr
ansf
orm
ed c
ells
wer
e m
appe
d by
2D
PA
GE
and
vis
uali
zed
by C
oom
assi
e B
lue
R25
0 st
aini
ng o
r 35
S/an
tora
diog
raph
y. P
rote
ins
of in
tere
st w
ere
iden
tifi
ed b
y E
dman
seq
uenc
ing
or b
y co
mpa
riso
n ag
ains
t pub
lish
ed 2
D m
aps.
Pro
tein
dis
ulfi
de is
omer
ase
(PD
I) w
as i
dent
ifie
d as
a p
oten
tial
mar
ker
for
tran
sfor
mat
ion.
Pro
tein
s w
hich
cop
reci
pita
ted
wit
h PD
I w
ere
also
iden
tifi
ed a
nd c
hara
cter
ized
. T
hese
resu
lts
wil
l be
disc
usse
d w
ithi
n th
e co
ntex
t of
SHE
cel
l tra
nsfo
rmat
ibn.
> >
E1
CQNFORNATIONAL COa~PJ~RXSON IN THE SN~KB TOXXN FAMILY
Roberto J. Falkensteln, Mirtha J. Biscoglio de Jim~nez Bonino and Clara Pefla
Instltuto de Qulmica y Fisicoqu~mlca Biol6gicas
(UBA-CONICET),
Facultad de
Farmacia y Bioqu~mlca, Jun~n 956, 1113 Buenos Aires, Argentina
The aim of this work is to perform a theoretical study of the conformational
homology between several members of the polypeptldlc snake toxin family. In
spite of displaying a common three-dlmensional folding pattern - a compact
core rich in disulfide bridges, three loops and a triple stranded ~-sheet -
show various pharmacological activities.
We applied the method of Kubota et al.
[Biochim. Biophys. Acta, 701, 242-252,
(1982) ].
Consensus sequences were inferred on the basis of function, genus and
sequence homology. The method establishes the relationship between an amino
acid sequence and its native conformation in terms of correlation coefficients
<c(i,j)> and identifies homologous sequences which have homologous native
conformations.
When long ~-neurotoxins,
binding to nicotinic acetylcholine receptor,
were
compared, a high variability in the N-terminal loop was detected. On the other
hand, a high homology degree between the consensus sequences from genera NaJa
and Ophiophagus,
and Dendroaspls was observed even when a C(i,j)=0.8 was
selected. The same conclusion applies to comparisons between genera Astrotia
and Pelamls, and Bungarus. However,
some conformational differences
around
position 30 appear when the consensus
sequence from genus Lauticauda
is
included in the matrix.
A high homology degree
[C(i,j)=0.8]
was observed also in the comparison
between sequences from most members of citotoxln subgroups.
An unexpected high conformational similarity was found between k-neurotoxins
- binding to the neuronal nicotinic receptor - and long u-neurotoxins.
In
contrast,
long and short u-neurotoxins
sharing the same target, display a
lower similarity degree.
Summarizing, Kubota'smethod allowed us to obtain important conclusions about
conformational homology in the snake toxin family, even though many of its
members have not yet been crystallized.
F]
GI aOLUTION STRUCTURE OF A CYCLIC ~-NSHANALOCUE-INITIALI~4RSTODIES
Michael F. Giblin I,
Tuck C. Wong 2 and~Thomas P. Quinn I,
Departments
of iBiochemfstry and 2Chemistry, University of Missouri, Columbla,MO 65211
x-Melanocyte Stimulating Hormone (~-MSH) is a 13 amino acid peptlde hormone
that stimulates melanin biosynthesis in vertebrates.
Cyclic analogues of
~-MSH have been designed which exhibit increased bloactlvity in vitro~
One
of these analogues is a disulfide cycllzed decapeptide which includes a D-Phe
replacement at position 4.
It has been hypothesized that a reverse turn
could be important for hormone activity due to the superpotency of such
cyclic analogues.
In the present study,th~ solution structure of this substituted analogue
is being investigated using NMR techniques.
NeE measurements in 80%H20/20%D~O
indicate a relatively constrained region between residues 3-5 of the peptide~
with considerably more conformational freedom around the disulfide bridge and
in the three residue tail.
Temperature dependent chemical shift studies of
the ~mide protons indicate no involvement in intramolecular hydrogen bonds,
and =J~NH measurements coupled with the pattern of sequential and non-
sequential NOE's do not conform to the classical picture of the~-turn.
The solvent 2,2,2-Trifluoroethanol-d 3 (TFE) has been postulated to act
as a membrane mimic, and is known to favor ~-hellx formation in linear
peptides.
NeE data obtained in 70% TFE/30% H20 is currently being compared
with the earlier data to determine whether this solvent mixture has an
ordering effect on the peptide.
G2
IDE
NT
IFIC
AT
ION
O
F
MO
DIF
IED
PT
H-A
MIN
O
AC
IDS
IN P
RO
TE
IN
SEQ
UE
NC
E
AN
AL
YSI
S G
rego
ry A
. G
rant
and
Mar
k W
. Cra
nksh
aw
Ass
ocia
tion
of
Bio
mol
ecul
ar
Res
ourc
e F
acil
itie
s an
d W
ashi
ngto
n U
nive
rsit
y Sc
hool
of
M
edic
ine,
St.
Lou
is,
MO
631
10.
The
ide
ntif
icat
ion
of
amin
o ac
id r
esid
ues
in m
oder
n pr
otei
n se
quen
ce
anal
ysis
em
ploy
ing
auto
mat
ed E
dman
deg
rada
tion
is
depe
nden
t on
the
elu
tion
pos
itio
n of
the
PT
H-
amin
o ac
ids
on h
igh
pres
sure
liq
uid
chro
mat
ogra
phy
syst
ems.
T
his
me
tho
d
reli
es o
n
a co
mpa
riso
n of
the
mig
rati
on o
f th
e un
know
n P
TH
-am
ino
acid
wit
h th
at o
f re
fere
nce
stan
dard
s.
Thi
s is
rel
ativ
ely
stra
ight
forw
ard
for
the
gene
tica
lly
code
d am
ino
acid
s,
but
beco
mes
pr
oble
mat
ic w
hen
mod
ifie
d or
unu
sual
am
ino
acid
res
idue
s ar
e pr
esen
t in
the
sam
ple
bein
g se
quen
ced.
Sin
ce t
he m
etho
d do
es n
ot p
rovi
de a
dir
ect
iden
tifi
cati
on o
f th
e P
TH
-am
ino
acid
, ad
diti
onal
ana
lysi
s by
che
mic
al o
r ph
ysic
al m
eans
is
nece
ssar
y. H
owev
er,
it is
oft
en h
elpf
ul t
o ha
ve s
ome
know
ledg
e of
whe
re k
now
n m
odif
ied
PT
H-a
min
o ac
ids
elut
e in
the
se s
yste
ms,
Thi
s pr
ovid
es a
sta
rtin
g po
int
for
the
inve
stig
ator
and
pro
vide
s an
add
itio
nal
leve
l of
kno
wle
dge
upon
whi
ch t
o pr
ocee
d.
A c
ompi
lati
on o
f un
usua
l PT
H-a
min
o ac
ids
has
been
und
erta
ken
by t
he A
ssoc
iati
on o
f B
iom
olec
ular
Res
ourc
e Fa
cilit
ies
(AB
RF
) in
an
atte
mpt
to
cons
olid
ate
info
rmat
ion
of t
his
type
fo
r ea
sy r
efer
ence
. It
sho
uld
be n
oted
tha
t an
exh
aust
ive
revi
ew o
f th
e lit
erat
ure
has
not
been
at
tem
pted
and
it h
as n
ot b
een
poss
ible
to
inde
pend
entl
y ve
rify
the
info
rmat
ion
pres
ente
d he
re.
Rat
her,
mem
bers
of
the
AB
RF
wer
e as
ked
to s
ubm
it a
ny in
form
atio
n th
ey h
ad in
thi
s re
gard
for
incl
usio
n in
thi
s bo
okle
t. W
hen
liter
atur
e re
fere
nces
hav
e be
en p
rovi
ded
they
are
inc
lude
d~
The
refo
re,
this
inf
orm
atio
n is
int
ende
d to
be
used
onl
y as
a g
uide
. A
ddit
iona
l su
ppor
ting
an
alys
es s
houl
d be
em
ploy
ed t
o ve
rify
the
iden
tity
of
any
unko
wn
resi
due.
Thi
s co
mpi
lati
on i
s in
tend
ed f
or u
se b
y th
e en
tire
sci
enti
fic
com
mun
ity
and
is a
vail
able
to
any
one
who
is
inte
rest
ed.
G3
CH
AR
AC
TER
IZA
TIO
N
OF
RE
CO
MB
INA
NT
PO
RIN
BY
MA
SS
SP
EC
TRO
ME
TRY
F. G
uine
t 1 , Y
. Pe
tilto
t 2, J
.M.
Cha
psal
3,
J. D
ubay
le I
, F.
Gre
co 1
, O.
Bar
ge 3,
E.
Fore
st 2,
C
. Va
lent
in I
Mas
s m
easu
rem
ent
of a
re
com
bina
nt
pori
n of
M
r =
4547
0 w
as
perf
orm
ed
on
an
elec
tros
pray
mas
s sp
ectr
omet
er (
Perk
in E
lmer
Sci
ex A
PIII)
. Th
is m
embr
ane
prot
ein
was
po
orly
sol
uble
in
solv
ents
and
buf
fers
(cl
assi
caly
use
d fo
r m
ass
spec
trom
etry
), an
d w
e pe
rfor
med
the
ana
lysi
s in
pur
e fo
rmic
aci
d. P
erfo
rman
t sp
ectr
a w
ere
obta
ined
, ho
wev
er
we
obse
rved
a r
apid
evo
lutio
n of
the
spe
ctra
und
er s
tora
ge i
n fo
rmic
aci
d. T
he i
ncre
ase
of m
ass
obse
rved
cou
ld b
e du
e to
for
myl
atio
ns.
Oth
er c
ondi
tions
of
solu
biliz
atio
n ha
ve
been
set
whi
ch g
aran
tee
any
poly
pept
ide
chan
ge.
Usi
ng t
his
tech
niqu
e w
e an
alys
ed
seve
ral
batc
hes
of th
is p
rote
in,
and
we
saw
mas
s m
odifi
catio
ns f
or c
erta
in p
repa
ratio
ns.
Ana
lysi
s (b
y se
quen
cing
an
d m
ass
dete
rmin
atio
n)
of
the
pept
ide
map
of
th
ose
poly
pept
ides
(e
nzym
atic
fin
qer
prin
ting)
al
low
ed
us t
o ge
t a
bett
er u
nder
stan
ding
of
th
ose
mod
ifica
tions
mai
nly
due
to C
-ter
min
al d
egra
datio
n.
1 R
esea
rch
and
Dev
elop
men
t D
epar
tmen
t/Pr
otei
n Q
ualit
y C
ontr
ol
Pas
teur
Med
eux
S~ru
ms
de V
acci
ns,
Fran
ce
2 La
bora
toir
e de
Spe
ctro
met
rie
de M
asse
des
Pro
tein
es
Inst
itut d
e B
iolo
gie
Stru
ctur
ale,
Gre
nobl
e, F
ranc
e 3
Res
earc
h an
d D
evel
opm
ent
Depa
rtmen
t~Ba
cter
iolo
gy
Pas
teur
Mer
ieux
Ser
ums
de V
acci
ns,
Fran
ce
G5
t~
CRYSTALLIZATION OF A THERMALSTABLETRYPSIN I~IBITORFROMP~SEOLUS
LUNATUS
Scott Griffith, Steve Schroeder, Thomas Quinn
Department of Biochemistry, University of Missouri, Columbia, MO 65211
In contrast to all other known Kunitz-type trypsin inhibitors, lima beans
contain a Kunitz-type trypsin inhibitor which can be subjected to high
temperatures for a significant time without destroying its ability to
actigely inhibit trypsin (i). Due to this attribute, Lima Bean Trypsin
Inhibitor (LBTI) has been made the subject of a structural investigation
as a m
odel of protein thermal stability.
Crude LBTI extract was obtained
and purified by an initial heat treatment followed by affinity chromatography.
The thermal stability of LBTI was examined by a trypsin activity assay.
Pure
LBTI and Soybean Trypsin Inhibitor (STI) were heated from 85~
to 92~
over
15 m
inutes.
The LBTI retained its activity while STI lost its ability to
actively inhibit trypsin.
Circular Diehroism thermal melt data suggests
similar LBTI secondary structure at 25 ~ and 80~
Two crystallization
conditions identified by sparse-matrix sampling were optimized, yielding
cubic crystals with dimensions of 0.5 mm.
Initial diffraction studies
have shown diffraction to a resolution of 2.7 Angstroms.
Future investigation
will reveal the tertiary structure of this protein and provide insight on
the physical nature of LBTI thermal stability.
Supported by Hughes
Undergraduate Research Internship.
Reference:
Tauber, H., Kershaw, B.B., and Wright R.D. (1949) J. Biol. Chem. 17___99:1155
G4
Insights into the Structure and Active-Site Architecture of IPP:DMAPP
Isomerase
Frederick M Hahn, Jonathan A. Baker and C. Dale Poulter,
University of
Utah
Genetic and biochemical methods have allowed for the further
characterization
of the enzymic structural elements that facilitate
the essential interconversion
of isopentenyl diphosphate and
dimethylallyl
diphosphate,
Isolation of the S. pombe isomerase gene
was performed by a plasmid shuffle mediated complementation strategy.
Amino acid sequence alignment from three isomerase sources allowed for
the identification of possible active sight regions in the proteins
primary structure and identified 52 n-terminal amino acids of the S.
cerevisiae protein as catalytically non-essential.
The lack of
catalytic importance for these n-terminal amino acids was confirmed by
complementation analysis with a truncated S. cerevisaie isomerase
gene.
A high yield overexpression
and purification protocol has been
developed for recombinant wild type and mutant S. pombe isomerases
that has greatly facilitated the crystallization
of the enzyme for x-
ray crystallographic
analysis.
Two amino acids that are beleived to
be important in the protonation/deprotonation
steps of the enzymatic
reaction have been mutated and characterized.
These studies support
the putative roles of Cys87 and Glu152 as important catalytic
components of the S. pombe isomerase active site.
HI
rj~
> > P'
I
CO
UN
TIN
G C
YS
TE
INE
AN
D C
YS
TIN
E R
ES
IDU
ES
IN
PR
OT
EIN
S A
ND
PE
PT
IDE
S
US
ING
MA
LD
I-T
OF
MA
SS
SP
EC
TR
OM
ET
RY
Tor
ben
Hal
kier
& A
rne
Age
rlin
Ols
en,
Prot
ein
Cha
ract
eriz
atio
n an
d E
nzym
e Pu
rifi
cati
on,
Prot
ein
Che
mis
try,
In
dust
ial
Bio
tech
nolo
gy,
Bio
indu
stri
al G
roup
, N
ovo
Nor
disk
A
/S,
Den
mar
k.
The
num
ber
of c
yste
ine
and
cyst
ine
resi
dues
in
prot
ein
and
pept
ides
can
be
dete
rmin
ed in
a
vari
ety
of w
ays
of w
hich
com
plet
e se
quen
ce d
eter
min
atio
n by
pro
tein
seq
uenc
ing
or c
DN
A
sequ
enci
ng is
the
mos
t pre
cise
. V
ario
us t
itra
tion
met
hods
exi
st a
s w
ell.
We
have
use
d co
vale
nt m
odif
icat
ion
of p
rote
ins
and
pept
ides
com
bine
d w
ith
MA
LD
I-T
OF
m
ass
spec
trom
etry
to
deve
lop
easy
met
hods
for
cou
ntin
g of
cys
tein
e an
d cy
stin
e re
sidu
es i
n pr
otei
ns a
nd p
epti
des.
In
addi
tion
, w
e ha
ve b
een
able
to
dete
ct n
on-c
oval
ent c
ompl
exes
of
som
e pr
otei
ns w
ith
met
al i
ons.
We
wil
l di
scus
s co
rrel
atio
ns b
etw
een
enzy
me
stru
ctur
e an
d fu
ncti
on in
thi
s co
ntex
t.
H2
MLr
rAG
ENES
IS O
F TH
E C
YC
LIC
AM
P R
ECEP
TOR
PR
OT
EIN
(CR
P) O
F ES
CH
ERIC
HIA
CO
Ll.
Jam
es G
. H
arm
an,
Eun
Ju
Lee
, Jo
el G
lasg
ow,
Sew
Fen
Lew
an
d A
ll O
. B
eldu
z r~
D
ept.
of
Che
m.
and
Bio
chem
., T
exas
Tec
h U
niv.
, L
ubbo
ck,
TX
794
09
;}~
Ana
lysi
s of
the
CR
P:c
AM
P
crys
tal
stru
ctur
e id
enti
fied
am
ino
ac
id-l
igan
d ~"
co
ntac
ts c
onsi
dere
d im
po
rtan
t in
cA
MP
med
iate
d a
ctiv
atio
n of
CR
P (
1).
To
test
th
ese
pre
dic
tio
ns
CR
P w
as m
uta
gen
ized
to
subs
titu
te s
elec
ted
amin
o ac
ids
for
E72
, R
82,
$83,
T12
7 an
d $
128
(2).
T
he r
esul
ts o
f th
is s
tudy
con
firm
s a
role
for
bo
th E
72
and
R82
in
cAM
P b
indi
ng a
nd
act
ivat
ion
of C
RP
(2)
. In
con
tras
t, c
AM
P i
nter
acti
ons
wit
h $
83,
T12
7 an
d $
128
cont
ribu
te t
o cA
MP
-med
iate
d C
RP
act
ivat
ion
and
hav
e li
ttle
eff
ect
on
cA
MP
bin
ding
. T
he e
ffec
ts o
f am
ino
acid
su
bsti
tuti
ons
on
CR
P
func
tion
wer
e as
sess
ed i
n ce
lls
that
con
tain
ed t
he m
uta
nt
form
s of
CR
P a
nd
th
rou
gh
in
vit
ro l
ac t
rans
crip
tion
exp
erim
ents
. C
RP
aff
init
y fo
r li
gand
was
cha
ract
eriz
ed b
y m
easu
rem
ent
of c
AM
P b
ind
ing
to
puri
fied
wil
d-ty
pe a
nd
mu
tan
t C
RP
pre
para
tion
s.
Eff
ects
on
CR
P s
truc
ture
wer
e as
sess
ed b
yp
rote
ase
sens
itiv
ity
and
DT
NB
-med
iate
d in
ters
ubun
it c
ross
link
ing.
S
ubst
itut
ion
of T
127
by C
, G
, I,
or
S pr
oduc
e fo
rms
of C
RP
th
at d
emon
stra
te s
truc
tura
l ch
arac
teri
stic
s, i
n th
e ab
senc
e of
cA
MP
, si
mil
ar t
o th
ose
of t
he w
ild-
type
CR
P:c
AM
P c
ompl
ex.
Inte
ract
ions
inv
olvi
ng b
oth
the
met
hy
l g
rou
p
and
the
hyd
roxy
l g
rou
p o
f T
127
app
ear
to p
lay
an i
mp
ort
ant
role
sta
bili
zing
CR
P
stru
ctur
e m
the
abs
ence
of
cAM
P.
~Dis
rupt
ion
of t
hese
int
erac
tion
s an
d s
tabi
liza
tion
of
(an
) al
tern
ate
stru
ctur
e(s)
is
trig
gere
d ei
ther
by
cA
MP
bin
ding
or
by a
min
o a
cid
subs
titu
tion
of
T12
7.
We
pred
ict
that
j~r
ecis
e re
posi
tion
ing
of t
he T
127
side
cha
in i
s re
quir
ed f
or C
RP
act
ivat
ion
and
is
crxt
ical
to
the
tran
sfer
of
sign
al f
rom
th
e cy
clic
nu
cleo
tide
bin
ding
poc
ket.
1.
Web
er,
I. T
. an
d S
teit
z, T
. A
. (1
987)
J. M
ol.
Bio
l., 1
98,
311-
326.
2.
B
eldu
z, A
. O
., L
ee, E
. J.
and
Har
man
, J.
G.
(199
3) N
ucle
ic A
cids
Res
., 21
, 18
27-1
835.
H
4
D~
ON
O
F D
I-P
TH
-CY
S F
OR
AS
SIG
NM
EN
T O
F D
ISU
LF
IDE
L
INK
AG
ES
Mit
suru
H
aniu
, W
illi
am
C.
Ken
ney,
an
d M
icha
el
F.
Roh
de
Am
gen,
Inc
., T
hous
and
Oak
s,
CA
.
US
A
9132
0
Dip
hen
ylt
hio
hy
dan
toin
de
riva
tive
of
cy
stin
e w
as
sig
nif
ican
tly
re
cove
red
by
gas
phas
e E
dm
an
reac
tion
, w
hen
cy
stin
e li
nkag
e w
as
loca
ted
at
the
sam
e po
siti
on
in
two
pept
ides
. U
po
n
dire
ct
sequ
ence
an
alys
is
of
inta
ct
bo
vin
e in
suli
n,
the
diP
TH
-Cys
w
as
rele
ased
af
ter
cycl
e 7,
co
rres
pond
ing
to
a di
sulf
ide
link
age
Cys
-A7-
Cys
-B7,
at
a
leve
l of
ap
prox
imat
ely
20%
re
cove
ry
rela
tive
to
th
e co
mm
on
P
TH
- am
ino
acid
s.
How
ever
, th
e di
PT
H-C
ys
was
po
orly
re
cove
red
afte
r E
dm
an
cycl
e 11
, co
rres
pond
ing
to
Cy
s-A
6-C
ys-
All
li
nkag
e.
For
as
sig
nm
ent
of
disu
lfid
e li
nkag
es
in
pept
ides
w
ith
mor
e th
an
two
disu
lfid
e li
nkag
es,
sele
cted
pr
oteo
lysi
s m
ay
be
used
to
po
siti
on
the
pred
icte
d cy
stin
e fo
r di
PT
H-C
ys
dete
ctio
n.
Thi
s m
eth
od
w
as
appl
ied
to
seve
ral
pept
ide
frag
men
ts
of
PD
GF
fo
r de
term
inat
ion
of
com
plic
ated
di
sulf
ide
stru
ctur
e.
Sta
bili
ty
of
the
diP
TH
-Cy
s an
d th
e b
ack
gro
un
d
pro
ble
ms
are
disc
usse
d.
H3
EV
AL
UA
TIN
G P
RO
TE
IN M
OD
IFIC
AT
ION
SIT
ES
OB
SE
RV
ED
BY
N-T
ER
MIN
AL
SE
QU
EN
CE
AN
AL
YS
IS.
Ree
d J.
Har
ris.
G
enen
tech
, Inc
., So
. San
Fra
ncis
co,
CA
940
80.
Man
y s
ites
of
pro
tein
mod
ific
atio
n ar
e fi
rst
dete
cted
(pa
rtic
ular
ly i
n ne
wly
-
isol
ated
pro
tein
s) b
y th
e ap
pear
ance
of
an u
nu
sual
PT
H-a
min
o ac
id,
low
rec
over
y
of a
co
mm
on
PT
H-a
min
o ac
id,
or b
y th
e ap
pear
ance
of
a '"
olan
k" s
eque
ncin
g cy
cle.
The
inv
esti
gato
r is
th
en l
eft
to a
sk "
Wha
t w
as t
hat?
".
Bla
nk c
ycle
s m
ay b
e d
ue
to
N-g
lyco
syla
tion
, m
ost
typ
es o
f O
-gly
cosy
lati
on,
thio
este
rs,
~-h
yd
rox
yA
sp/A
sn,
eth
ano
lam
ine
ph
osp
ho
gly
cer0
1
moi
etie
s (o
n G
lu),
an
d
~-(y
-glu
tam
yl)-
Iysi
ne,
disu
lfid
e or
lan
thio
nine
cro
ss-l
inks
. L
ow r
ecov
erie
s of
co
mm
on
PT
H-a
min
o ac
ids
aris
ing
from
mod
ifie
d am
ino
acid
s in
clud
e y-
carb
oxyg
luta
mat
e an
d ph
osph
oser
ine.
Sta
ble
PT
H-d
eriv
ativ
es
are
ob
tain
ed
for
hy
dro
xy
lysi
ne,
h
yd
rox
yp
roli
ne
and
met
hy
late
d H
is,
Lys
, A
rg,
Gln
. G
lcN
Ac-
Asn
, fu
cosy
lthr
eoni
ne,
and
Gal
NA
c-
Ser
/Th
r ar
e pa
rtia
lly
reco
vere
d.
Isoa
spar
tate
cau
ses
an a
bru
pt
loss
of
sign
al.
Man
y
of
thes
e m
od
ific
atio
ns
hav
e co
nse
nsu
s se
qu
ence
s th
at
can
faci
lita
te
thei
r
iden
tifi
cati
on.
Obs
erve
d se
quen
ces
shou
ld b
e co
nfir
med
by
mas
s sp
ectr
omet
ry t
o
rule
out
the
pre
senc
e of
a l
abil
e m
odif
icat
ion
such
as
phos
phor
ylat
ion
or a
cyla
tion
.
H5
DE
TE
CT
ION
OF
LO
W L
EV
EL
S O
F H
YD
RO
XY
LY
SIN
E I
N N
ON
-CO
LL
AG
EN
OU
S
PRO
TE
INS.
Ree
d J.
Har
ris,
Mic
hael
S.
Mol
ony,
Len
e H
. K
eyt,
Shi
aw-L
in W
u.
Gen
ente
ch,
Inc.
,
460
Poi
nt S
an B
runo
Blv
d., S
o. S
an F
ranc
isco
, CA
940
80.
5-H
ydro
xyly
sine
(H
yl)
is a
n es
sent
ial p
ostt
rans
lati
onal
mod
ific
atio
n of
col
lage
ns
and
col
lage
n-li
ke r
egio
ns o
f ce
rtai
n pr
otei
ns.
Hyl
is
fo
un
d
in -
Xaa
-Hyl
-Gly
- se
quen
ces
and
may
be
furt
her
mod
ifie
d by
gly
cosy
lati
on.
We
have
ide
ntif
ied
low
leve
ls o
f H
yl (
0.1-
0.3
mo
l/m
ol
prot
ein)
at
sing
le s
ites
in
seve
ral
non-
coll
agne
ous
reco
mbi
nant
pro
tein
s is
olat
ed f
rom
tra
nsfe
cted
CH
O c
ell l
ines
, inc
ludi
ng L
ys-2
77 o
f rt
PA
and
Lys
-46
of r
CD
4 an
d rC
D4-
1gG
. H
um
an t
PA
fro
m t
he B
owes
mel
anom
a
cell
lin
e an
d a
tra
nsfe
cted
293
cel
l li
ne a
lso
show
low
lev
els
of H
yl-2
77.
Sen
siti
ve
dete
ctio
n of
Hyl
by
amin
o ac
id a
naly
sis
requ
ired
a m
odif
ied
LiC
itra
te-b
uffe
red
syst
em.
Seq
uenc
ing
yiel
ds o
f P
TH
-(e-
PT
C)-
Hyl
are
mu
ch l
ower
th
an f
or P
TH
-(e-
PTC
)-L
ys.
Hyl
sit
es w
ere
foun
d in
pep
tide
s w
here
exp
ecte
d ly
syl
bond
s w
ent
uncl
eave
d by
try
psin
, sug
gest
ing
that
Hyl
is
a po
or t
ryps
in s
ubst
rate
. T
he o
bser
ved
Hyl
Sit
es a
re i
n -X
aa-H
yl-G
ly-
sequ
ence
s w
ithi
n su
rfac
e-ac
cess
ible
reg
ions
.
H6
CH
EM
ICA
L E
XA
MIN
AT
ION
OF
TH
E D
IPH
EN
YL
PHO
SPH
OR
O-
ISO
TH
IOC
YA
NA
TID
AT
E/T
RIM
ET
HY
LSI
LA
NO
LA
TE
ME
TH
OD
FO
R P
RO
TE
IN
CA
RB
OX
YL
-TE
RM
INA
L D
EG
RA
DA
TIO
NS.
Dav
id H
. H
awke
, Ja
quel
ine
Tso
, Sh
erre
ll E
arly
and
Cha
d G
. M
ille
r.
Prot
ein
Che
mis
try
Syst
ems,
Hew
lett
-Pac
kard
Co.
, Pa
lo A
lto,
CA
94
304
USA
In th
e pa
st f
ew y
ears
a n
umbe
r of
reag
ents
hav
e be
en in
vest
igat
ed fo
r us
e in
car
boxy
te
rmin
al s
eque
nce
anal
ysis
via
thio
hyda
ntoi
n de
grad
atio
n.
Exa
mpl
es in
clud
e th
iocy
anat
e sa
lts,
thio
cyan
ic a
cid,
dip
heny
lpho
spho
rois
othi
ocya
nati
date
(DPP
-1T
C),
trim
ethy
lsily
lisot
hioc
yana
te,
trib
utyl
tin
isot
hioc
yana
te,
and
benz
oyl i
soth
iocy
anat
e.
Bai
ley
and
cow
orke
rs (
Prot
ein
Scie
nce(
1992
) 1,
162
2-16
33)
repo
rted
tha
t the
pre
viou
sly
know
n D
PP-I
TC
(K
enne
r et
al.,
J.
Che
m.
Soc.
, 19
53,
673-
678)
und
er n
ew c
ondi
tion
s al
low
ed d
egra
dati
on a
nd id
enti
fica
tion
of
all c
omm
on a
min
o ac
ids
exce
pt p
roli
ne.
Rel
ativ
e re
acti
on k
inet
ics
wer
e in
ferr
ed f
rom
pro
duct
dis
trib
utio
ns fo
r a
vari
ety
of
alte
rnat
e re
agen
t con
cent
rati
ons,
del
iver
y m
odes
, re
acti
on ti
mes
and
tem
pera
ture
s in
a
prot
ein
mod
el s
yste
m.
For
exa
mpl
e, i
n th
e or
igin
al re
port
s D
PP-I
TC
was
use
d as
a 3
M
solu
tion.
B
ased
on
our
reac
tion
stu
dies
1M
is a
suf
fici
entl
y hi
gh c
once
ntra
tion
to a
ssur
e go
od c
oupl
ing,
but
at
low
er c
once
ntra
tion
s the
rea
ctio
n is
com
prom
ised
. R
esul
ts a
re
pres
ente
d w
ith
emph
asis
on
deve
lopm
enta
l pat
hs f
or r
eact
ion
opti
miz
atio
n.
H7
ATPASE AND MOLECULAR CHAPERONE ACTIVITIES OF THE MAIZE
ENDOPLASMIC RETICULUM-RESIDENT STRESS-70-PROTEIN
G. Thomas Hayman and Jan A. Miernyk
USDA, ARS, NCAUR,
Phytoproducts Research Unit,
1815 N.
University Street,
Peoria,
IL 61604
Members of the 70 kDa family of stress-related proteins
(Stress-70)
function as molecular chaperones.
Distinct forms
of Stress-70 are present within all subcellular compartments
of eukaryotic cells.
Stress-70s have a low level endogenous
ATPase activity that is stimulated several-fold by short
peptides of undefined sequence.
The importance of ATPase
activity in molecular chaperone function is a point of debate.
To clarify the importance of ATPase activity, we have
undertaken mutagenesis of a maize ER-resident Stress-70 cDNA.
Native and mutant proteins have been produced in E.
coli as
MBP-fusions,
purified by affinity chromatography,
and the MBP
sequence removed by specific proteolysis.
Native Stress-70
binds to immobilized ATP and can be specifically displaced by
free ATP.
A mutant Stress-70 lacking a putative ATP-binding
domain does not bind appreciably to immobilized ATP.
The
results of other functional analyses of native and mutant
Stress-70 will be presented.
H8 M
ICR
OPR
EPA
RA
TIO
N F
OR
SE
QU
EN
CE
AN
AL
YSI
S O
F P
EPT
IDE
S O
F P
RO
TE
INS
OB
TA
INE
D F
RO
M O
LD
AR
CH
IVE
D P
OL
YA
CR
YL
AM
IDE
GE
LS
BY
IN S
ITU
T
RY
PSIN
DIG
EST
ION
.
Utf
HeU
man
, Chr
iste
r Wem
sted
t and
Jorg
e G
6fie
z L
udw
ig I
nsti
tute
for C
ance
r Res
earc
h, B
ox 5
95, S
-751
24
Upp
sala
, Sw
eden
We
have
impr
oved
a p
ublis
hed
proc
edur
e fo
r ge
nera
tion
of
inte
rnal
pro
tein
fra
gmen
ts b
y in
ge
l tr
ypsi
n di
gest
ion
of s
ampl
e, s fr
eshl
y is
olat
ed b
y SD
S-PA
GE
(1)
. O
ur m
odif
icat
ion,
whi
ch
invo
lves
com
plet
e dr
ying
of
the
gel
prio
r to
adm
inis
trat
ion
of t
he p
rote
ase,
has
giv
en t
he
proc
edur
e a
mor
e ge
nera
l ap
plic
abili
ty;
we
now
ro
utin
ely
anal
yze
prot
eins
fr
om
poly
acry
lam
ide
gels
whi
ch h
ad b
een
run
for
anal
ytic
al p
urpo
se,
Coo
mas
sie
stai
ned,
dri
ed f
or
arch
ivin
g an
d st
ored
for
var
ying
len
gths
of
time.
Aft
er in
situ
dig
esti
on a
nd e
xtra
ctio
n,
the
gene
rate
d fr
agm
ents
axe
sep
arat
ed b
y re
vers
ed p
hase
liq
uid
chro
mat
ogra
phy
in o
ne o
r tw
o di
men
sion
s on
a
SMA
RT
Sy
stem
an
d su
bjec
ted
to
sequ
ence
an
alys
is
on
an A
pplie
d B
iosy
stem
s M
odel
470
A i
nstr
umen
t. T
he p
roce
dure
wor
ks w
ell
wit
h sa
mpl
es i
n th
e 10
-20
pmol
e ra
nge,
and
it a
ppea
rs t
hat t
he s
tora
ge o
f pro
tein
ban
ds in
the
drie
d ge
ls,
eith
er o
n fi
lter
pape
r or
bet
wee
n pl
astic
wra
p, d
oes
no h
arm
to
the
prot
ein.
E
ven
tryp
toph
an r
esid
ues,
no
tori
ousl
y kn
own
for t
heir
sens
itivi
ty to
bec
ome
degr
aded
hav
e be
en p
ositi
vely
ide
ntif
ied.
1. R
osen
feld
, J.,
Cap
devi
elle
, J.,
Gui
llem
ot, J
. C.,
and
Ferr
ara,
P. (
1992
) A
nal.
Bio
chem
. 20
3,
173-
179.
H9
g
IDE
NTI
FIC
ATI
ON
AN
D C
HA
RA
CTE
RIZ
ATI
ON
O
F M
ET
AL
PR
OTE
IN C
OM
PLE
XE
S O
F
CR
UD
E C
ELL
EX
TRA
CTS
B
Y M
AS
S-S
PE
CT
RO
ME
TR
Y
Dan
iel H
ess,
Ral
ph S
tude
r an
d P
eter
E.
Hun
zike
r B
ioch
emis
ches
Ins
titut
de
r U
nive
rsit~
it Zt
'iric
h, W
inth
ertu
rers
tr.
190,
CH
-805
7 Z
firic
h,
Sw
itzer
land
We
repo
rt
the
use
of
a m
icro
bo
re
reve
rse-
phas
e hi
gh
pe
rfo
rma
nce
liq
uid
chro
mat
ogra
phy
syst
em c
onne
cted
on
line
to a
n el
ectr
ospr
ay m
ass
spec
trom
eter
for
the
sepa
ratio
n an
d ch
arac
teri
zatio
n of
int
act p
rote
in-m
etal
com
plex
es o
f va
riou
s fo
rms
of m
etal
loth
ione
in is
olat
ed f
rom
diff
eren
t hu
man
cel
l lin
es.
App
roxi
mat
ely
10%
of
the
colu
mn
efflu
ent
was
div
erte
d th
roug
h a
flow
-spl
ittin
g de
vice
into
the
ion
sou
rce
of th
e m
ass
spec
trom
eter
, w
here
as t
he m
ajor
ity o
f th
e m
ater
ial
wa
s co
llect
ed.
Thi
s fir
st
anal
ysis
was
per
form
ed a
t pH
6.0
and
the
mol
ecul
ar w
eigh
ts o
f th
e in
tact
com
plex
es
wer
e de
term
ined
. Sin
ce t
he m
etal
ions
are
rel
ease
d fr
om m
etal
loth
ione
in a
t lo
w p
H,
a se
cond
ana
lysi
s w
as c
arri
ed o
ut o
n th
e m
etal
free
apo
prot
eins
aft
er a
cidi
ficat
ion
of t
he
isol
ated
met
al c
ompl
exes
. F
rom
the
mas
s di
ffer
ence
s ob
tain
ed,
accu
rate
met
al t
o pr
otei
n st
ochi
omet
ries
cou
ld b
e ca
lcul
ated
. The
det
ectio
n lim
it of
ind
ivid
ual i
sofo
rms
of
met
allo
thio
nein
der
ived
fro
m a
tot
al o
f 10
x 1
06 c
ells
(2.
5mg
of t
otal
cel
lula
r pr
otei
n)
was
abo
ut 3
0pm
ol.
The
met
hod
desc
ribe
d is
use
d to
rap
idly
iden
tify
diff
eren
t is
ofor
ms
of m
etal
ioth
ione
in
in
crud
e ce
ll ex
trac
ts.
Add
ition
ally
, it
.allo
ws
the
accu
rate
det
erm
inat
ion
of h
ow m
any
and
whi
ch m
etal
ions
are
inco
rpor
ated
into
the
diffe
rent
isof
orm
s.
HIO
TH
E S
TRU
CTU
RE
AN
D F
UN
CTI
ON
OF
PLA
NT
LEG
INS
ULI
N
His
ash
i H
ira
no
, Y
osh
ihir
o
Wa
tan
ab
e 1
, S
erg
ei
F.
Ba
rba
sho
v 1,
S
ets
uko
K
om
ats
u 1
, A
nd
rew
M.
He
mm
ing
s 1,
Mas
aru
Miy
agi 2
and
Sus
umu
Tsu
na
saw
a 2
Nat
l. In
st.
Ser
icul
t.
Ent
omot
. S
ci.,
1N
atl.
Inst
. A
gro
bio
l.
Re
sou
rce
s,
Tsu
kuba
, Ib
arak
i, Ja
pan
and
2Bio
tech
. R
es,
Lab.
, T
akar
a S
huzo
, O
tsu,
Shi
ga,
Japa
n.
A 4
kD
a p
ep
tid
e n
am
ed
te
gins
ulin
, w
hich
ca
n bi
nd
to
the
soyb
ea
n
ba
sic
7S
glo
bu
lin
(an
insu
lin-b
ind
ing
p
rote
in
in
pla
nts
) a
nd
co
mp
ete
w
ith
in
sulin
fo
r bi
ndin
g to
th
e
ba
sic
7S
glob
ulin
w
as
isol
ated
fr
om
the
so
ybe
an
se
eds.
T
he
com
ple
te
am
ino
a
cid
se
qu
en
ce
of
the
le
gin
sulin
w
as
de
term
ine
d
by
au
tom
ate
d
Ed
ma
n
de
gra
da
tio
n
and
ele
ctro
spra
y io
niz
ati
on
m
ass
sp
ect
ro-
met
ry.
It co
nsi
ste
d
of
37
am
ino
ac
id
resi
du
es
wit
h
6 h
alf
-cys
tin
es
in
3 d
isu
lfid
e
brid
ges.
T
he
m
ass
sp
ect
rom
etr
ic
an
aly
sis
reve
ale
d
tha
t a
port
ion
of t
he p
ep
tid
e i
s p
roce
sse
d t
o d
ele
te t
he
C-t
erm
ina
l g
lyci
ne
lik
e a
nu
mb
er
of
an
ima
l p
ep
tid
e
ho
rmo
ne
s,
bu
t no
t C
-te
rmin
all
y a
mid
ate
d.
Th
e
cDN
A
enco
ding
the
leg
insu
lin w
as
clon
ed,
seq
ue
nce
d a
nd
co
nsi
de
red
to
cod
e f
or a
p
recu
rso
r p
oly
pe
pti
de
co
nsi
stin
g
of
a p
uta
tiu
e
sig
na
l p
ep
tid
e,
the
le
g-
insu
lin,
a lin
ker
pe
pti
de
, a
6 kD
a
pe
pti
de
and
a
C-t
erm
ina
l p
ep
tid
e.
Th
e
legi
nsul
in
had
a st
imu
lato
ry
effe
ct
on
the
a
ctiv
ity
of
pro
tein
ki
na
se
of
the
b
asi
c 7S
g
lob
ulin
, su
gg
est
ing
th
at
the
le
gin
sulin
is
in
volv
ed
in
ce
llula
r si
ng
na
l tr
an
sdu
ctio
n.
Th
ere
w
as
no
seq
ue
nce
h
om
olo
gy
be
twe
en
th
e
leg-
in
sulin
an
d in
sulin
o
r in
sulin
-lik
e
gro
wth
fa
cto
rs.
Hll
PRO
BIN
G T
HE
Mg 2
+ B
IND
ING
SIT
E O
F B
-GA
LA
CT
OSI
DA
SE (E
. coi
l )
Reu
ben
E.
Hub
er,
Nat
han
J. R
oth,
Mic
hael
T.
Gau
nt
Div
isio
n of
Bio
chem
istr
y, F
acul
ty o
f Sci
ence
, Uni
vers
ity
of C
alga
ry, C
alga
ry,
Can
ada
T2N
1N
4
Mg 2
+ ac
tiva
tes
wil
d-ty
pe 8
-gal
acto
sida
se (E
. ce
ll ).
The
Mg 2
+ bi
ndin
g si
te o
f 13
-gal
acto
sida
se o
f E
. co
il
is m
ade
up o
f G
lu-4
61,
His
-418
, an
d G
lu-4
16 (
1,2,
3).
We
have
mad
e si
te-d
irec
ted
subs
titu
tion
s fo
r th
ese
thre
e re
sidu
es a
nd s
tudi
ed th
e co
nseq
uenc
es o
n th
e ki
neti
cs a
nd t
he m
etal
bi
ndin
g.
Eac
h su
bsti
tuti
on f
or G
lu-4
61 t
hat
we
trie
d ca
used
the
los
s of
a l
arge
am
ount
of
the
acti
vity
and
the
enz
yme
beca
me
inac
tiva
ted
rath
er t
han
acti
vate
d w
hen
Mg 2
+ w
as a
dded
. T
his
inac
tiva
tion
was
slo
w a
nd h
ighl
y pH
dep
ende
nt.
Whe
n su
bsti
tute
d by
His
, th
e en
zym
e be
cam
e br
oadl
y sp
ecif
ic fo
r di
vale
nt m
etal
s bu
t it b
ound
Mg 2
+ re
lati
vely
poo
rly.
Sub
stit
utio
n of
a P
be f
or
His
-418
cau
sed
the
enzy
me
to b
ind
Mg 2
+ ve
ry p
oorl
y. S
ubst
itut
ion
wit
h a
Glu
res
tore
d th
e M
g 2+
bind
ing
but a
gain
, w
hen
Mg 2
+ w
as b
ound
, the
enz
yme
was
inac
tiva
ted.
Su
bsti
tuti
on o
f H
is-4
18
by A
sn r
esul
ted
in a
n en
zym
e w
hich
bou
nd M
g 2+
poor
ly,
but
in t
hat
case
, ad
diti
on o
f M
g 2+
ac
tiva
ted
the
enzy
me.
Sub
stit
utio
n fo
r G
lu-4
16 r
esul
ted
in a
n en
zym
e w
ith
poor
Mg 2
+ bi
ndin
g ab
ilit
y bu
t whe
n M
g 2+
was
add
ed, t
he e
nzym
e lo
st a
ctiv
ity.
The
res
ults
indi
cate
that
sub
stit
utio
ns
for e
ach
of th
e th
ree
Mg 2
+ li
gand
s at
the
acti
ve s
ite
of 1
3-ga
lact
osid
ase
caus
e th
e en
zym
e to
hav
e lo
wer
ed a
ctiv
ity
wit
h re
spec
t to
Mg 2
+.
The
eff
ect t
hat
Mg 2
+ ha
s is
ver
y se
nsit
ive
to c
hang
es o
f th
e li
gand
and
the
effe
ct o
f M
g 2+
on th
e ac
tivi
ty is
usu
ally
the
reve
rse
of th
e ef
fect
of M
g 2+
on th
e ac
tivi
ty o
f th
e no
rmal
enz
yme.
1.
R
.A.
Edw
ards
et a
l.,
Bio
chem
. B
ioph
ys. R
es~
Com
m.
171,
33
(199
0)
2.
N.J
. R
oth
and
R.E
. H
uber
, B
ioch
em. B
ioph
ys. R
es.
Com
m.
In P
ress
(19
94)
3.
R. J
acob
sen
and
B. M
atth
ews,
Per
sona
l Com
mun
icat
ion
H1
2
IN-G
EL
R
ED
UC
TIO
N
AN
D
AL
KY
LA
TIO
N
OF
P
RO
TE
INS
A
ND
E
NZ
YM
AT
IC
DIG
ES
TIO
N:
AP
PL
ICA
TIO
N
TO
T
HE
S
EQ
UE
NC
E
AN
AL
YS
IS
OF
P
RO
TE
INS
S
EP
AR
AT
ED
BY
1-D
GE
L E
LE
CT
RO
PH
OR
ES
IS
Pau
l Je
n6,
Thi
'err
y M
ini,
Suz
ette
MoB
s, M
arti
n H
orst
, D
epar
tmen
t of
Bio
chem
istr
y,
Bio
zent
rum
of
the
Uni
vers
ity
of B
asel
, K
ling
elbe
rgst
rass
e 70
, 40
56 B
asel
, S
wit
zerl
and.
In r
ecen
t ye
ars,
alt
erna
tive
s to
the
blo
ttin
g te
chni
ques
, su
ch a
s in
-sit
u di
gest
ion
of
prot
eins
in
th
e po
lyac
ryla
mid
e m
atri
x w
ere
esta
blis
hed.
W
hen
w
e ap
plie
d th
ese
proc
edur
es t
o st
and
ard
pro
tein
s su
ch a
s ly
sozy
me
and
RN
A's
e A
we
foun
d th
at n
o
usef
ul d
iges
ts c
ould
be
obta
ined
, re
gard
less
wh
eth
er t
he p
rote
ins
wer
e di
gest
ed i
n th
e ge
l m
atri
x or
fro
m P
VD
F m
embr
anes
. H
ow
ever
, co
mpl
ete
dige
st i
n ge
l pi
eces
wer
e ob
tain
ed w
hen
th
ey w
ere
redu
ced
and
carb
oxym
ethy
late
d pr
ior
to e
lect
roph
ores
is.
The
se r
esul
ts i
ndic
ate
that
, al
thou
gh p
rote
ins
are
red
uce
d i
n th
e L
aem
mli
sam
ple
buf
fer,
on
ce t
he r
edu
cin
g a
gent
is
rem
ov
ed d
uri
ng
ele
ctro
phor
esis
, th
ey a
re a
ble
to f
old
into
co
mpa
ct d
isul
fide
-lin
ked
stru
ctur
es w
hic
h a
re d
iffi
cult
to
dige
st.
We
ther
efor
e de
vise
d an
in-
situ
red
ucti
on a
nd a
lkyl
atio
n pr
oced
ure
for
prot
eins
con
fine
d in
gel
pie
ces.
In
shor
t, p
rote
ins
are
redu
ced
in 0
,1 M
Tri
s-H
CL
pH
8.0
, 0.
1% S
DS,
10
mM
DT
T a
t 60
oC
for
45 m
in.,
fol
low
ed b
y a
lkyl
atio
n w
ith
50
mM
iod
oace
tam
ide.
Dig
esti
on i
s d
on
e w
ith
A
chro
mob
acte
r pr
otea
se I
. P
rior
to
chro
no
mat
og
rap
hy
on
nar
row
-bor
e re
vers
e-ph
ase
colu
mns
, th
e b
ulk
SD
S i
s re
mov
ed w
ith
1 M
gua
nidi
nium
-chl
orid
e. T
he p
roce
du
re w
as
succ
essf
ully
app
lied
to
the
sequ
ence
det
erm
inat
ion
of v
ario
us i
sofo
rms
of N
-ace
tyl
tran
sfer
ase
fro
m D
roso
phila
m
elan
ogas
ter
and
for
a n
um
ber
of
yeas
t m
itoc
hond
rial
inn
er
mem
bra
ne
prot
eins
fro
m 2
-dim
ensi
onal
O'F
arre
ll g
els.
J1
BI
AN
AL
YS
IS O
F T
HE
EX
TR
AC
EL
LU
LA
R O
RG
AN
IZA
TIO
N O
F T
HE
a-C
HA
IN O
F
MO
US
E A
CE
TY
LC
HO
LIN
E R
EC
EP
TO
R O
N L
IVE
MU
SC
LE
CE
LL
S
Ken
ji J
inna
i 1'2
, Tet
suo
Ash
izaw
a 1 a
nd
M.
Zo
uh
air
Ata
ssi 2
D
epar
tmen
ts o
f N
euro
l. 1
and
Bio
chem
. ,
Bay
lor
Col
lege
of
Med
icin
e, H
ou
sto
n,
TX
A c
om
pre
hen
siv
e sy
nthe
tic
stra
tegy
(1)
was
em
plo
yed
to
synt
hesi
ze a
pan
el o
f 16
- o
r 17
- re
sidu
e pe
ptid
es,
whi
ch o
ver
lap
ped
con
secu
tive
ly b
y 5
resi
du
es a
nd e
nco
mp
asse
d t
he
enti
re m
ain
ext
race
llul
ar p
art
of
the
a-ch
ain
of
mo
use
nic
otin
ic a
cety
lcho
line
rec
epto
r (m
AC
hR
).
Th
e bi
ndin
g o
f an
tib
od
ies
agai
nst
each
of
the
pep
tid
es t
o m
AC
hR
on
mo
use
sk
elet
al m
usc
le c
ells
in
cult
ure
rev
eale
d t
he
extr
acel
lula
r o
rgan
izat
ion
of
the
a-ch
ain
in
//re
mus
cle
cell
s an
d in
dica
ted
that
th
e re
gio
ns
a23-
49,
a78-
126,
a1
46-1
74
and
a18
2-
210
are
acce
ssib
le t
o bi
ndin
g w
ith
the
resp
ecti
ve a
nti-
pept
ide
anti
bodi
es.
Co
mp
aris
on
o
f th
is b
indi
ng p
rofi
le w
ith
that
rec
entl
y re
po
rted
(2)
fo
r T
orpe
do
cali
forn
ica
AC
hR
in
isol
ated
mem
bra
ne
frac
tion
s sh
ow
ed t
hat
reg
ion
s a8
9-10
4 an
d a
158-
174
rem
ain
ex
po
sed
ac
ross
the
spe
cies
in l
ive
cell
s an
d in
iso
late
d m
emb
ran
e fr
acti
ons.
Th
ere
wer
e, h
owev
er,
sign
ific
ant
diff
eren
ces
in t
he e
xtra
cell
ular
org
aniz
atio
n w
ithi
n th
ese
two
env
iro
nm
ents
. R
esid
ues
a23-
49,
a78-
126
and
a194
-210
hav
e b
een
sh
ow
n t
o co
nta
in r
egio
ns
that
are
in
volv
ed i
n th
e bi
ndin
g o
f a-
neu
roto
xin
s (3
-5)
and
of
auto
anti
bo
die
s in
so
me
my
asth
enia
gr
avis
(M
G)
pat
ien
ts (
6).
Th
e ex
po
sed
reg
ion
s de
fine
d by
thi
s st
udy
may
th
us
be
targ
ets
for
the
init
ial
auto
imm
un
e at
tack
on
the
rec
epto
r in
MG
.
(1)
Kaz
im, A
.L. a
nd A
tass
i, M
.Z. (
1980
) Bio
chem
. J.
191
, 261
-264
; (2)
Ata
ssi,
M.Z
. and
Mul
ac-J
eric
evlc
, B.
(199
4) J.
Pro
t. C
hem
. 13
, 37
-48;
(3)
Mul
ac-J
eric
evic
, B. a
nd A
tass
i, M
.Z. (
1987
) Bio
chem
. Z
248
, 847
-852
; (4
) R
uan,
K.H
. eta
l., (
1990
) Pro
c. N
atl.
Aca
d. S
ci.
USA
87,
615
6-61
60; (
5) R
uan,
K.H
. eta
l., (
1991
) Bio
chem
. J.
274
, 849
-854
; (6)
A
shlz
awa,
T. e
t al.,
(19
92) M
oL I
mm
unol
. 29
, 15
07-1
514.
J2
SEPA
RA
TIO
N
AN
D
CH
AR
AC
TE
RIZ
AT
ION
O
F P
RO
TE
INS
WIT
H
TW
O
DIM
EN
SIO
NA
L
EL
EC
TR
OPH
OR
ESI
S
Mas
ahan
l Kam
o, T
akao
Kaw
akam
i, Nor
ifum
i Miy
atak
e and
Aki
ra T
sugi
ta
Res
earc
h Ins
titut
e for
Bio
scie
nces
, Sci
ence
Uni
vers
ity of
Tok
yo, Y
amaz
aki 2
669,
Nod
a, C
hiba
278,
Japa
n.
Hig
h re
solu
tion t
wo
dim
ensi
onal
poly
acry
lam
ide g
el e
lect
roph
omsi
s (2-
DE
) is
one
of th
e m
ost a
dvan
ced
tech
niqu
es
used
to p
rovi
de se
para
tion o
f con
side
rabl
e num
bers
of p
rote
ins.
The
met
hod p
erm
its th
e re
solu
tion o
f aro
und 5
,000
pr
otei
ns o
n on
e pl
ate (
22.5
x 2
2.5
cm).
Int
rodu
ctio
n of t
he im
mob
ilize
d pH
gra
dien
t str
ip al
low
s a re
prod
ucib
le an
d w
ide
rang
e pH
(pH
3-pH
l0) g
radi
ent f
or th
e fi
rst d
imen
sion
of
2-D
E.
Dev
elop
men
t of
com
pute
r sof
twar
e for
sc
anni
ng, i
nteg
ratio
n, q
uant
itativ
e an
alys
is an
d st
orag
e of
the
info
rmat
ion h
as r
emar
kabl
y co
ntri
bute
d on
2-D
E
tech
nolo
gy.
Fart
her,
dire
ct m
icro
-seq
uenc
e ana
lysi
s of t
he a
min
o(N
)-te
rmin
al pa
rtia
l seq
uenc
e of t
he e
lect
robl
otte
d pr
otei
n sp
ots
from
the
gels
and
the
hom
olog
y sea
rch
agai
nst P
IR-I
nter
natio
nal p
rote
in se
quen
ce d
atab
ase
have
pr
ovid
ed id
entif
icat
ion
of th
e pr
otei
ns in
add
ition
to th
e co
nven
tiona
l ide
ntif
icat
ion
proc
esse
s suc
h as
com
igra
tion.
W
e di
scus
s on
the
resu
lts o
f 2-D
E o
f bot
h ri
ce p
rote
ins(
l) a
nd A
rabi
dops
is pr
otei
ns an
d se
vera
l nov
el te
chni
ques
in
clud
ing
com
igra
tion w
ith ex
tern
al st
anda
rd m
arke
r pro
tein
s for
stan
dard
izat
ion
and
editi
on o
f 2-D
E g
el p
atte
rns a
nd
nove
l deb
lock
ing
met
hods
of N
-ter
min
al b
lock
ages
of p
rote
ins w
ith a
nhyd
rous
hydr
azin
e vap
or(2
).
Nin
e tis
sues
(le
af, s
tem
, roo
t, ge
rm, s
eedl
ing,
seed
, bra
n, ch
aff a
nd c
allu
s) an
d on
e or
gane
lle (c
hlor
opla
st) w
ere u
sed f
or th
e in
tegr
al
rice
pro
tein
s. F
or e
ach
rice
tiss
ues,
abou
t 250
-150
0 pr
otei
n sp
ots w
ere s
epar
ated
on a
2-D
E g
el.
By
the
use
of th
e co
mig
rate
d ni
ne m
arke
r pro
tein
s(3)
, the
se im
ages
wer
e edi
ted
into
one
imag
e whi
ch re
sulte
d in
4,8
92 p
rote
in sp
ots.
W
e so
far u
sed
only
a fi
xed g
el c
once
ntra
tion
12.5
% w
hich
may
loos
e the
reso
lutio
n for
pro
tein
s les
s tha
n 12
kD
a an
d hi
gher
than
70 k
Da.
Fro
m th
ese s
pots
140
prot
eins
(2.9
%) w
ere a
naly
zed a
nd 5
6 pr
otei
ns (1
.1%
) hav
e bee
n se
quen
ced
but t
he o
ther
84 p
rote
ins w
ere b
lock
ed at
the
N-t
erm
ini (
60%
). F
rom
Ara
bido
psis
, 5 ti
ssue
s wer
e ana
lyze
d for
thei
r pr
otei
ns an
d 4,
760
prot
ein
spot
s wer
e ide
ntif
ied.
Fif
ty-t
wo s
pots
wer
e ele
ctro
blot
ted
and
sequ
ence
d. E
ight
een s
pots
w
ere i
dent
ified
from
the
sequ
ence
hom
olog
y with
PIR
sequ
ence
data
base
. The
oth
er te
n sp
ots w
ere s
eque
nced
but n
ot
yet i
dent
ified
. T
he o
ther
twen
ty fo
ur sp
ots h
ave t
he b
lock
ed N
-term
ini.
In A
rabi
dops
is pr
otei
ns w
e fou
nd al
so ab
out
60%
blo
cked
N-t
erm
ini o
f th
e to
tal
prot
eins
test
ed.
Com
bina
tion 2
DE
with
pre
-fra
ctio
natio
n by
bio
logi
cal
prop
ertie
s, su
ch as
aff
inity
chro
mat
ogra
m w
ith D
NA
-col
umn o
r fer
redo
xin c
olum
n may
hel
p th
e ch
arac
teriz
atio
n of
pr
otei
ns w
ith th
e pa
rtial
pro
tein
sequ
ence
s or t
he o
pen r
eadi
ng fr
ame s
eque
nces
. 1.
A. T
sugi
ta et
al.,
Ele
ctro
phor
esis
(in
pres
s).
2. N
. Miy
atak
e et a
l., E
ur. J
. Bio
chem
., 212
, 785
(!9
93).
3. R
.J. S
imps
on et
al.,
Ele
ctro
phor
esis
, 13,
105
5 (1
992)
. K
1
PR
OB
ING
OF
TH
E P
HY
LO
GE
NY
OF
TH
E C
CK
/GA
ST
RIN
FA
MIL
Y B
Y A
NT
ISE
RA
D
IRE
CT
ED
AG
AIN
ST
TH
E C
ON
SE
RV
ED
CO
MM
ON
C-T
ER
MIN
US
.
An
der
s H
. Jo
hn
sen
, H
ann
e Je
nse
n,
Jen
s F
. R
ehfe
ld
Dep
artm
ent
of
Clin
ical
B
ioch
emis
try,
Rig
shos
pita
let,
Uni
vers
ity
of
Co
pen
hag
en,
Den
mar
k.
Th
e n
euro
end
ocr
ine
pept
ides
, C
CK
(ch
olec
ysto
kini
n) a
nd
gas
trin
, or
igin
ally
ide
ntif
ied
in
mam
mal
s, c
onst
itut
e a
fam
ily
shar
ing
the
C-t
erm
inus
, T
rp-M
et-A
sp-P
he
�9 NH
2, w
hic
h i
s al
so
the
min
imal
str
uct
ure
nec
essa
ry f
or b
iolo
gica
l ac
tivity
. T
he
iden
tity
of
the
acti
ve s
ite
sugg
ests
th
at C
CK
an
d g
astr
in h
ave
evol
ved
fro
m a
co
mm
on
an
cest
or
wit
h an
ap
par
entl
y h
ighl
y co
nser
ved
C-t
erm
inus
. U
sin
g a
nti
sera
dir
ecte
d ag
ains
t th
e co
mm
on
C-t
erm
inu
s, C
CK
-lik
e im
mun
orea
ctiv
ity
has
bee
n o
bse
rved
in
mo
st v
erte
bra
te a
nd
in
ver
teb
rate
phy
la.
In o
rder
to
inve
stig
ate
the
phyl
ogen
y o
f th
e C
CK
/gas
trin
fam
ily
we
have
use
d s
uch
anti
sera
to
mo
nit
or
the
puri
fica
tion
of b
oth
en
do
crin
e an
d n
euro
pep
tid
es f
rom
spe
cies
rep
rese
nti
ng
sev
eral
phy
la
thro
ug
ho
ut
the
anim
al k
ingd
om.
As
rep
rese
nta
tiv
es o
f in
ver
teb
rate
pep
tid
es a
cco
un
tin
g f
or
CC
K-l
ike
imm
unor
eact
ivit
y th
e ly
mn
aDF
amid
es w
ere
isol
ated
fro
m s
nail
gan
glia
. Th
ey s
har
e th
e tw
o C
-ter
min
al r
esid
ues
wit
h C
CK
/gas
trin
sug
gest
ing
that
no
rea
l C
CK
pep
tid
es e
xist
in
inve
rteb
rate
s, b
ut
they
may
all
bel
on
g to
an
Asp
-Ph
e-am
ide
supe
rfam
ily.
Ide
ntif
icat
ion
of f
ish
CC
K's
and
of
the
pro
toch
ord
ean
cio
nin
sho
w th
at C
CK
/gas
trin
pep
tid
es c
an b
e d
ated
mo
re
than
500
mil
lion
yea
rs b
ack.
Bir
ds,
rept
iles
, an
d am
ph
ibia
con
tain
sep
arat
e C
CK
an
d g
astr
in
pep
tid
es s
ho
win
g t
hat
thei
r di
verg
ence
occ
urr
ed b
efo
re t
he e
volu
tion
of
amp
hib
ia.
Th
e C
CK
's f
rom
am
ph
ibia
an
d u
pw
ard
are
dea
rly
evo
luti
onar
y re
late
d. S
imil
arit
ies
of
fro
g-C
CK
an
d -
gast
rin
sup
po
rt t
he h
yp
oth
esis
of
thei
r di
verg
ence
fro
m a
co
mm
on
anc
esto
r. H
ow
ever
, m
amm
alia
n g
astr
ins
diff
er s
o m
uch
tha
t ye
t an
oth
er g
ene
dupl
icat
ion
mu
st b
e su
gg
este
d t
o ac
coun
t fo
r th
eir
evol
utio
n.
J3
AN
IN
VE
ST
IGA
TIO
N O
F T
HE
BA
TH
OC
HR
OM
IC
SH
IFT
PH
EN
OM
EN
ON
IN
B
IOL
OG
Y
JN K
een,
P
F
Zag
alsk
y I
& J
BC
Fin
dla
y D
ept B
ioch
emis
try
& M
olec
ular
Bio
logy
, Uni
vers
ity o
f L
eeds
, L
eeds
, L
S2
9JT
, U
K a
nd 1
Dep
t B
ioch
emis
try,
Roy
al H
ollo
way
, U
nive
rsit
y of
Lon
don,
Egh
am,
Sur
rey,
TW
20 0
EX
, U
K.
The
bat
hoch
rom
ic s
pect
ral
shif
t ph
enom
enon
, ce
ntra
l to
the
pro
cess
of
visi
on i
n bo
th
vert
ebra
te a
nd i
nver
tebr
ate
syst
ems,
has
yet
to
be p
rope
rly
expl
aine
d w
ith
resp
ect
to t
he
mec
hani
sm o
f in
volv
emen
t of
the
prot
ein
com
pone
nt o
f th
e rh
odop
sin-
reti
nal
com
plex
. T
he
use
of
com
pute
rgra
phic
mod
elli
ng h
as p
rovi
ded
muc
h us
eful
inf
orm
atio
n on
the
put
ativ
e st
ruct
ure
of
rhod
opsi
n, b
ut a
def
init
ive
crys
tal
stru
ctur
e ha
s ye
t to
con
firm
the
fea
ture
s co
nsid
ered
impo
rtan
t in
func
tion.
P
aral
l~ to
our
inve
stig
atio
n of
str
uctu
re-f
unct
ion
rela
tion
ship
s in
rho
dops
in, w
e ha
ve b
een
stud
ying
the
str
uctu
res
of
a n
um
ber
of
othe
r pr
otei
ns w
hich
bin
d si
mil
ar p
olye
ne-c
onta
inin
g m
olec
ules
and
und
ergo
alm
ost i
dent
ical
bat
hoch
rom
ic s
pect
ral s
hift
s.
The
fir
st tw
o of
thes
e (t
he c
rust
acya
nin
subu
nits
A 2
and
CI)
hav
e be
en f
ully
seq
uenc
ed (
1,
2) a
nd t
heir
str
uctu
res
mod
elle
d on
the
hom
olog
ous
retin
ol-b
indi
ng
prot
ein.
X
-ray
ana
lysi
s of
cr
ysta
ls is
wel
l und
erw
ay.
Fur
ther
pro
tein
s ar
e al
mos
t com
plet
ely
sequ
ence
d.
The
aim
of
this
app
roac
h is
to
lear
n su
ffic
ient
abo
ut t
he s
truc
ture
of
caro
teno
id b
indi
ng
pock
ets
in g
ener
al,
in a
n at
tem
pt t
o un
ders
tand
ho
w th
e ba
thoc
hrom
ic s
hift
is
brou
ght a
bout
. S
uch
know
ledg
e m
ay th
en b
e ap
plic
able
to
rhod
opsi
n.
We
pres
ent h
ere
the
sequ
ence
of
the
caro
teno
id-b
indi
ng
prot
ein
linck
iacy
anin
, fr
om th
e sk
in
of
the
star
fish
Lin
ckia
la
evig
ata,
a
glyc
opro
tein
of
appr
oxim
atel
y 10
kD
a, w
hich
ass
embl
es in
to
helic
al a
rray
s of
ove
r lx
l06
Da.
1.
J.N
. K
een
et
al.,
Eu
r. J
. B
ioch
em.,
197
, 40
7 (1
991)
. 2.
J.
N.
Kee
n e
t al
., E
ur.
J.
Bio
chem
., 2
02,
31 (
1991
).
1(2
MIC
RO
SEQ
UE
NC
ING
O
F PR
OT
EIN
S O
F T
HE
E
ND
OPL
ASM
IC
RE
TIC
UL
UM
(E
R)
ME
MB
RA
NE
Reg
ine
Kra
ft 1,
2, S
usan
ne K
ostk
a 1,2
, Enn
o H
artm
ann 1
1M
ax-D
elbr
tick-
Cen
ter
for
Mol
ecul
ar M
edic
ine,
Ber
lin-B
uch,
Ger
man
y an
d 2H
umbo
ldt U
nive
rsit
y,
Ber
lin, G
erm
any
The
E
R
mem
bran
e is
an
im
port
ant
orga
nelle
in
volv
ed
in
such
div
erse
fun
ctio
ns a
s pr
otei
n tr
ansl
ocat
ion,
pr
otei
n fo
ldin
g an
d ph
osph
olip
id
bios
ynth
esis
. O
nly
few
pro
tein
s ha
ve s
o fa
r be
en
iden
tifie
d (s
ee f
or i
nsta
nce
1, 2
). W
e ha
ve s
tart
ed a
sys
tem
atic
ana
lysi
s of
thes
e pr
otei
ns b
y us
ing
a m
ini-
two-
dim
ensi
onal
(2D
) PA
GE
tec
hniq
ue,
follo
wed
by
blo
tting
of
the
sep
arat
ed
pept
ides
ont
o PV
DF
mem
bran
es an
d au
tom
ated
sequ
enci
ng.
Her
e w
e pr
esen
t res
ults
from
the
N-t
erm
inal
am
ino
acid
seq
uenc
ing
of tw
enty
one
pro
tein
spo
ts fr
om
one
to f
our
Coo
mas
sie
Blu
e st
aine
d PV
DF
mem
bran
es.
Com
pari
son
of t
he a
min
o ac
id s
eque
nces
ob
tain
ed w
as c
arri
ed o
ut u
sing
the
FAST
A c
ompu
ter p
rogr
am o
f the
Gen
etic
s Com
pute
r Gro
up.
Seve
n pr
otei
n sp
ots
coul
d be
cle
arly
ide
ntif
ied.
Fro
m th
e re
mai
ning
one
s in
suff
icie
nt se
quen
ce in
form
atio
n w
as a
vaila
ble
or th
e N
-ter
min
us w
as b
lock
ed.
The
pre
limin
ary
frac
tiona
tion
of th
e w
hole
mic
roso
mal
pr
epar
atio
n by
det
erge
nt p
artit
ioni
ng (
Tri
ton
X-1
14)
into
lum
inal
and
mem
bran
e fr
acti
ons
prov
ided
ad
ditio
nal
conc
lusi
ons
abou
t th
e ce
llula
r lo
caliz
atio
n of
the
poly
pept
ides
ide
ntif
ied
and
shou
ld a
lso
resu
lt in
an
incr
ease
d re
solu
tion
of th
e in
divi
dual
pol
ypep
tide
spot
s.
l.
T. R
apop
ort
et a
l., A
nton
ie v
an L
eeuw
enho
ek,
61, 1
19 (1
992)
. 2.
E
. Har
tman
n et
al.,
Eur
. J. B
ioch
em.,
214,
375
(19
93).
K3
DETERMINATION
OF
THE
PRIMARY
STRUCTURE
OF
A NEW
MILK-CLOTTING
PROTEASE BY MICROSEQUENCING
TECHNIQUES
Claudia Machalinski,
Mirtha Blscoglio
de Jim~nez Bonino
Institute
of
Biological
Chemistry
and
Phlslcochemistry
(CONICET),
University
of
Buenos
Aires,
Junin
956,
(1113)
Buenos
Aires,
Argentina
Microsequeneing
techniques
were
applied
in order
to
determine
the
moiecular
architecture
of
a milk
clotting
protease
from
Mucor
bacilliformis
(I).
In order
to localize
the disulfide
bridges,
both the native and the
reduced
and
4-vinyl
piridine-treated
p~oteases
were
digested
with
trypsin
and
the
resulting
peptide
mixtures
were
submitted
to
RP-
HPLC;
the elutions
were monitored
at 220,
254 and 278 nm.
Peptides
which
absorbing
at
254
nm
were
sequenced.On
the
other
hand,
structural
analyses
were
performed
with
the
PVDF-elecroblotted
protein:
a)
direct
N-sequencing
determination
allowed
obtention
of
the
sequence
of
the
first
33
protease
aminoaeld
residues,
b)
cyanogen-bromide
treatment
of the native
as well
as of the reduced
and vinyl
pirldlne-treated
protein
rendered
two
internal
sequences
in addition
to that
of
the
N-
terminal
fragment,
c)
by using
the
method
of
Wadsworth
(reaction
with
o-phthalaldehyde)-
leading
to
the
isolation
of
a unique
sequence
from
a polypeptide
mixture-
a long important
internal
sequence was obtained.
Summarizing,
these
mlcrosequencing
techniques
led
to
the
determination
of
structural
characteristiques
of
a local
protease
whose
biological
behavior
could
make
it
a good
substitute
for
bovine chymosin
in cheese manufacture.
(I) Purification
and Characterization
of a
Milk Clotting
Protease
from Mucor bacilliformis.
L. B. Areees,
M. Biscoglio
de Jim~nez
Bonino,
M. A. A. Parry,
E. Fraile,
H. M. Fernandez
and O. Caseone.
M1
>.
ml
N-I
SO
PR
OP
YL
IOD
OA
CE
TA
MID
E I
N T
HE
RE
DU
CT
ION
AN
D A
LK
YL
AT
ION
OF
P
RO
TE
INS:
U
SE
IN
MIC
RO
SE
QU
EN
CE
AN
AL
YS
IS
Hen
ry C
. K
rutz
sch
and
John
K.
Inm
an 1
Lab
orat
ory
of P
atho
logy
, Nat
iona
l C
ance
r In
stit
ute,
NIH
, B
ethe
sda,
MD
and
l L
abor
ator
y of
Im
mun
olog
y, N
atio
nal
Inst
itut
e o
f A
ller
gy a
nd I
nfec
tiou
s D
isea
ses,
NIH
, B
ethe
sda,
MD
A n
ew r
eage
nt, N
-iso
prop
ylio
doac
etam
ide
(NIP
IA),
for
alk
ylat
ion
of
sulf
hydr
yl g
roup
s on
pr
otei
ns f
or m
icro
dige
stio
n an
d m
icro
sequ
enci
ng is
des
crib
ed.
The
uti
lity
of
this
rea
gent
in
bot
h o
f th
ese
proc
edur
es h
as b
een
dem
onst
rate
d. N
IPIA
was
sho
wn
to b
e es
peci
ally
us
eful
in
mic
rose
quen
ce a
naly
sis,
whe
re i
t yi
elds
hig
h se
nsit
ivit
y in
det
ecti
on
of C
ys
resi
dues
. T
his
is b
ecau
se t
he p
heny
lthi
ohyd
anto
in (
PT
H)
deri
vati
ve o
f N
IPIA
-aik
ylat
ed
cyst
eine
[P
TH
-Cys
(NIP
CA
M)]
app
ears
as
a sh
arp
peak
in
a st
anda
rd r
ever
se-p
hase
HP
LC
an
alys
is o
f P
TH
am
ino
acid
s, a
nd e
lute
s be
twee
n P
TH
-Tyr
and
PT
H-P
ro w
here
no
othe
r pe
aks
are
pres
ent.
Thu
s th
e us
e of
NIP
IA c
ircu
mve
nts
vari
ous
prob
lem
s as
soci
ated
wit
h H
PL
C
anal
ysis
of
PT
H-C
ys
whe
n ot
her
com
mon
ly u
sed
agen
ts
are
empl
oyed
fo
r su
lfhy
dryl
alk
ylat
ion,
suc
h as
co-
elut
ing
peak
s or
low
sig
nal
leve
ls.
Pro
cedu
res
for
the
synt
hesi
s of
NIP
IA a
nd o
ther
ana
logs
, as
wel
l as
PT
H-C
ys(N
IPC
AM
) ar
e al
so p
rese
nted
, an
d H
PL
C r
eten
tion
tim
es fo
r th
eir
corr
espo
ndin
g P
TH
-Cys
der
ivat
ives
are
com
pare
d.
K4
A p
H D
EP
EN
DE
NC
E O
N T
HE
AS
SO
CIA
TIO
N P
RO
PE
RT
IES
OF
MO
DE
L C
OIL
ED
CO
IL P
EP
TID
ES
Don
ald
K.
McR
orie
1, G
regg
R
. D
ieck
man
n 2,
Sus
an
Hei
lman
2, W
illia
m
F. D
eGra
do 3,
and
V
ince
nt
L.
eeco
raro
2 ~B
eckm
an I
nstru
men
ts,
Pal
o A
lto,
CA
, 2D
ept.
of C
hem
istr
y, U
niv.
of
Mic
h.,
Ann
Arb
or,
MI
and
3DuP
ont-
Mer
ck P
harm
aceu
tical
s,
Wilm
ingt
on,
DE
Coi
led
coil
sequ
ence
s re
port
ed in
a w
ide
vari
ety
of p
rote
ins
all c
onsi
st o
f hep
tad
amin
o ac
id r
epea
ts (
a b
c d
e f g
), w
ith h
ydro
phob
ic r
esid
ues
in t
he a
and
d p
ositi
ons.
T
his
posi
tioni
ng
resu
lts i
n th
e hy
drop
hobi
c re
sidu
es o
n on
e si
de o
f e
form
ed c
~-he
lix in
tera
ctin
g w
ith o
ther
hel
ices
with
the
sam
e st
ruct
ure.
The
str
uctu
re o
f tw
o sy
nthe
tic p
eptid
es w
ith th
is c
hara
cted
stic
hep
tad
repe
at s
eque
nce
wer
e ex
amin
ed b
y an
alyt
ical
ultr
acen
tdfu
gatio
n.
The
con
trol
pep
tide,
Td,
has
the
sequ
ence
Ac-
G(L
KA
LEE
K)4
G-N
H
2. A
sec
ond
pept
ide,
(T
ri(L1
2C))
, ha
s C
yste
ine
subs
titut
ed
for
Leuc
ina
in p
ositi
on
12.
We
are
inte
rest
ed
in d
esig
ning
m
etal
lope
ptid
es o
f kn
own
aggr
egat
ion
stat
e. S
ince
met
al b
indi
ng a
ffini
ty m
ay b
e de
pend
ent
on p
H,
we
ev
alua
ted
the
aggr
egat
ion
stat
e of
the
pep
tidas
un
der
diff
eren
t ac
idity
con
ditio
ns.
Due
to
the
lack
of
arom
atic
sid
e ch
ains
, th
e pe
ptid
e co
ncen
trat
ion
had
to b
e m
onito
red
in th
e fa
r uv
(230
nm)
whi
ch e
xclu
des
use
of s
ulfh
ydry
l re
agen
ts t
hat
abso
rb l
ight
at
this
wav
elen
gth.
At
pH 2
.5,
both
pep
tides
ha
d a
wei
ght
aver
age
mol
ecul
ar w
eigh
t (M
w)
of a
bout
700
0 w
hen
mod
eled
as
a si
ngle
id
eal
spec
ies
indi
catin
g th
e pr
edom
inan
ce o
f a d
imer
stru
ctur
e.
But
at p
H 8
.5,
both
pep
tides
had
an
Mw
clo
ser t
o tr
imer
. T
he g
reat
est
diff
eren
ce o
ccur
red
at p
H 5
.5 w
here
Td
was
clo
ser t
o th
e td
mer
Mw
obs
erve
d at
pH
8.5
and
Tri
(L12
C)
was
ap
prox
imat
ely
the
dim
er M
w o
bser
ved
at p
H 2
.5.
Nei
ther
pep
tide
fit w
ell
to t
he s
ingl
e sp
ecie
s m
odel
in
dica
ting
mor
e co
mpl
ex m
ixtu
res
of s
peci
es in
all
case
s.
Res
ults
are
con
sist
ent w
ith e
mod
el m
onom
er-d
imer
equ
ilibr
ium
at
pH
2.5
whi
ch c
hang
es t
o m
onom
er-
trim
er in
the
sim
ples
t m
odel
at
pH 8
.5.
The
Cys
sub
stitu
tion
wou
ld th
en a
ppar
ently
des
tabi
lize
the
trim
eric
st
ruct
ure
at p
H 5
.5 w
ith t
he c
ontr
ol a
lrea
dy c
hang
ing
to t
he t
dmer
equ
ilibr
ium
at
this
int
erm
edia
te p
H.
M2
C-T
ER
MIN
AL
PR
OT
EIN
SE
QU
EN
CE
AN
AL
YSI
S U
SIN
G T
HE
A
LT
ER
NA
TIV
E
RE
VE
RS
E-P
HA
SE
S
UP
PO
RT
S
FO
R D
IRE
CT
EL
EC
TR
OE
LU
TIO
N
t~
HE
WL
ET
T-P
AC
KA
RD
C
-TE
RM
INA
L P
RO
TE
IN S
EQ
UE
NC
ING
SY
STE
M
AN
D/N
SIT
U D
IGE
ST
ION
ON
TH
E H
EW
LE
TT
-PA
CK
AR
D S
EQ
UE
NC
ING
CO
LU
MN
.~
Cha
d G
. Mill
er, J
ames
Ken
ny, J
ulie
Sah
akia
n, D
avid
H. H
awke
, Ja
cque
line
Tso
Pr
otei
n C
hem
istr
y Sy
stem
s, H
ewle
tt-Pa
ckar
d C
o.,
Palo
Alto
, C
A 9
4304
An
auto
mat
ed C
-ter
min
al p
rote
in s
eque
ncin
g sy
stem
wit
h m
ultip
le s
ampl
e ca
pabi
lity
has
been
de
velo
ped
by th
e H
ewle
tt-Pa
ckar
d C
ompa
ny.
A n
ovel
seq
uenc
ing
chem
istr
y ba
sed
on C
ity o
f H
ope
tech
nolo
gy u
sing
dip
heny
lpho
spho
rois
othi
ocya
natid
ate
(DPP
-IT
C)
enab
les
the
sequ
entia
l deg
rada
tion
of C
-ter
min
al a
min
o ac
id r
esid
ues.
T
he s
eque
nce
anal
ysis
of p
rote
in
sam
ples
on
reac
tion
mem
bran
es (
Zit
ex) w
itho
ut p
re-s
eque
ncin
g co
vale
nt a
ttac
hmen
t che
mis
try
elim
inat
es in
itial
sam
ple
loss
es a
nd p
rovi
des
a co
nven
ient
bas
is f
or s
ampl
e lo
adin
g.
The
C
-ter
min
al s
eque
ncin
g m
etho
dolo
gy ru
ns a
t a 6
0 ra
in c
ycle
tim
e, h
as a
rep
rodu
cibl
e on
-lin
e th
iohy
dant
oin
amin
o ac
id s
tand
ard,
and
relia
ble
iden
tific
atio
n of
any
of t
he c
omm
on a
min
o ac
id
resi
dues
as
dem
onst
rate
d by
a b
road
ran
ge o
f div
erse
sam
ple
type
s.
The
met
hodo
logy
ena
bles
th
e se
quen
ce a
naly
sis t
hrou
gh p
rolin
e re
sidu
es y
ield
ing
thio
hyda
ntoi
n-pr
olin
e de
riva
tives
. D
ata
are
pres
ente
d de
mon
stra
ting
the
sequ
ence
ana
lysi
s of
pro
tein
s re
pres
entin
g a
rang
e of
mol
ecul
ar
wei
ghts
and
str
uctu
ral
com
plex
ity (
inte
rpre
ted
in te
rms
of re
acti
on e
ffic
ienc
ies
and
amin
o ac
id
back
grou
nd)
isol
ated
in
vari
ous
buff
er s
yste
ms
incl
udin
g cy
toch
rom
es,
lyso
zym
e, s
uper
oxid
e di
smut
ase,
hem
oglo
bins
, tr
iose
phos
phat
e is
omer
ase,
lact
oglo
bulin
, ser
um a
lbum
ins,
dis
solv
ed i
n aq
ueou
s sa
lt so
lutio
ns (
NaC
I, K
CI,
guan
idin
e) a
nd o
rgan
ic s
olve
nts
as H
PLC
fra
ctio
ns.
Met
hods
for
sequ
ence
ana
lyse
s of
sam
ples
blo
tted
from
SD
S ge
ls to
mem
bran
e su
ppor
ts
(Zite
x, P
VD
F) a
lso
exam
ined
.
M3
Mar
y B
. Moy
er a
nd W
illi
am A
. B
urkh
art
Gla
xo R
esea
rch
Inst
itut
e, R
esea
rch
Tri
angl
e P
ark,
NC
, 27
709,
US
A
Eff
icie
nt t
echn
ique
s fo
r sa
mpl
e pr
epar
atio
n us
ing
the
HP
seq
uenc
ing
colu
mn
pack
ed
wit
h a
C18
mat
eria
l w
ere
prev
ious
ly d
evel
oped
for
dir
ect
elec
troe
luti
on o
f pr
otei
ns
from
pol
yacr
ylam
ide
gels
an
d f
or i
n si
tu
dige
stio
n us
ing
a va
riet
y of
pro
teas
es.
How
ever
, hi
ghly
hy
dro
ph
ob
ic p
rote
ins
and
pept
ides
ca
n b
ind
irr
ever
sibl
y to
C18
su
ppor
ts.
Wh
en c
hara
cter
izin
g a
prot
ein,
it
is o
ften
nec
essa
ry t
o re
cove
r as
com
plet
e a
set
of p
epti
des
as p
ossi
ble.
W
e ha
ve,
ther
efor
es i
nves
tiga
ted
the
use
of t
he H
P
sequ
enci
ng c
olu
mn
pac
ked
wit
h P
OR
OS
R1
and
R2
perf
usiv
e su
ppor
ts (
Per
Sep
tive
B
iosy
stem
s) f
or u
se w
ith
hig
hly
hydr
opho
bic
prot
eins
. P
OR
OS
R1
in p
arti
cula
r ha
s sh
ow
n m
ark
ed i
mp
rov
emen
t in
rec
over
ies
of i
ntac
t hy
drop
hobi
c pr
otei
ns a
s w
ell
as
hydr
opho
bic
pept
ides
gen
erat
ed b
y in
sit
u di
gest
ion
usin
g ph
osph
odie
ster
ase
(55
kDa)
as
a m
odel
. T
his
capa
bili
ty a
llow
s th
e sa
mpl
e ha
ndli
ng t
echn
ique
s de
velo
ped
for
the
HP
seq
uenc
ing
colu
mn
to b
e us
ed f
or s
ampl
es a
naly
zed
by m
ass
spec
trom
etry
as
wel
l as
Ed
man
seq
uenc
ing.
In
a p
revi
ous
stud
y, t
he H
P C
18 c
olum
n w
as f
ound
to
be
inco
mpa
tibl
e w
ith
a c
apil
lary
PO
RO
S R
2 co
lum
n fo
r th
e is
olat
ion
of p
epti
des
gene
rate
d by
in
situ
dig
esti
on.
Dat
a w
ill
be s
ho
wn
dem
onst
rati
ng t
he s
ucce
ssfu
l co
upli
ng o
f th
e P
OR
OS
R1
colu
mn
foll
owin
g in
sit
u di
gest
ion
to a
cap
illa
ry P
OR
OS
R1
colu
mn
for
sepa
rati
on a
nd
dir
ect
coll
ecti
on o
f pe
ptid
es o
nto
Zit
ex m
embr
anes
.
M5
ESC
HE
RIC
HIA
CO
LI
DM
APP
-tR
NA
TR
AN
SFE
RA
SE
EX
PRE
SSIO
N A
ND
CH
AR
AC
TE
RIZ
AT
ION
Je
ffre
y A
. M
oore
and
C.
Dal
e Po
ulte
r. D
epar
tmen
t of
Che
mis
try,
Uni
vers
ity
of U
tah.
T
he m
odif
icat
ion
of n
ucle
osid
es
in t
RN
A c
onfe
rs u
niqu
e pr
oper
ties
to
the
mac
rom
olec
ule
impo
rtan
t to
its
biol
ogic
al fu
ncti
on.
A m
odif
icat
ion
obse
rved
in tR
NA
s of
bot
h eu
bact
eria
and
eu
kary
otes
is
the
isop
ente
nyla
tion
of
the
amin
o m
oiet
y of
ade
nosi
ne-3
7 ad
jace
nt to
ant
icod
ons
that
rea
d co
dons
beg
inni
ng w
ith
urid
ine.
T
he e
nzym
e re
spon
sibl
e fo
r th
is p
reny
l tr
ansf
er i
s di
met
hyla
llyl
diph
osph
ate:
tR
NA
di
met
hyla
llyl
tr
ansf
eras
e (D
MA
PP-t
RN
A
tran
sfer
ase)
. R
ecom
bina
nt E
. co
li
DM
APP
-tR
NA
tr
ansf
eras
e ha
s be
en o
vere
xpre
ssed
and
pur
ifie
d to
ap
pare
nt h
omog
enei
ty i
n tw
o st
eps
by i
on-e
xcha
nge
and
imm
unoa
ffin
ity
chro
mat
ogra
phy.
Pu
rifi
catio
n w
as g
reat
ly f
acil
itat
ed b
y th
e in
corp
orat
ion
of a
C-t
erm
inal
tri
pept
ide
a-tu
buli
n ep
itope
(gl
u-gl
u-ph
e) d
urin
g PC
R a
mpl
ific
atio
n. I
niti
al s
tudi
es w
ith
the
puri
fied
rec
ombi
nant
en
zym
e ha
ve s
how
n th
at th
e ac
tive
for
m o
f th
e pr
otei
n is
a m
onom
er,
and
that
the
DM
APP
- tR
NA
tra
nsfe
rase
-cat
alyz
ed
reac
tion
occ
urs
in a
tim
e an
d co
ncen
trat
ion
man
ner.
M4
A C
ON
VE
NIE
NT
P
RO
CE
DU
RE
F
OR
DE
TE
RM
INA
TIO
N
OF
CA
RB
OX
Y-
TE
RM
INA
L
AM
INO
A
CID
S
OF
P
RO
TE
INS
IM
MO
BIL
IZE
D
ON
T
HE
M
EM
BR
AN
E
Tat
yana
Mur
anov
a an
d L
ubov
Mak
ova
Bra
nch
of S
hem
yaki
n an
d O
vchi
nnik
ov I
nsti
tute
of
Bio
orga
nic
Che
mis
try,
Rus
sian
A
cade
my
of S
cien
ces,
Pus
hchi
no,
Mos
cow
Reg
ion,
142
292,
Rus
sia
A n
ovel
app
roac
h to
car
boxy
-ter
min
al s
eque
nce
anal
ysis
is
deve
lope
d.
The
cla
ssic
al
carb
oxyp
epti
dase
dig
esti
on is
sti
ll w
idel
y us
ed i
n th
is i
mpo
rtan
t are
a of
str
uctu
ral
stud
ies
of n
ew p
rote
ins.
T
he
maj
or d
isad
vant
age
of t
his
proc
edur
e is
to
be s
een
in t
he w
ell-
kn
own
fact
tha
t m
any
prot
eins
are
ins
olub
le u
nder
the
con
vent
iona
l enz
ymat
ic d
iges
tion
co
ndit
ions
and
, co
nseq
uent
ly,
are
indi
gest
ible
. In
the
new
ly d
evel
oped
met
hodo
logy
m
enti
oned
in
the
titl
e, t
he s
olub
ilit
y pr
oble
m h
as b
een
solv
ed v
ia i
mm
obil
izat
ion
of a
pr
otei
n on
the
Mil
lipo
re I
mm
obil
on-P
mem
bran
e.
Aft
er i
mm
obil
izat
ion
of a
pro
tein
sa
mpl
e,
the
resi
dual
fr
ee
prot
ein-
bind
ing
site
s of
the
m
embr
ane
are
bloc
ked
by
incu
bati
ng i
n th
e po
lyvi
nylp
yrro
lido
ne s
olut
ion.
T
he n
ext
step
is
carb
oxyp
epti
dase
Y
dige
stio
n of
mem
bran
e-bo
und
prot
ein.
A
liqu
otes
of
dige
st a
re w
ithd
raw
n at
zer
o ti
me
and
afte
r ap
prop
riat
e ti
me
inte
rval
s an
d ar
e an
alyz
ed d
irec
tly
for
free
am
ino
acid
s. T
he
impo
rtan
t ad
vant
age
of t
he n
ew a
ppro
ach
is i
ts i
ndep
ende
nce
from
pI
of a
pro
tein
. T
he m
etho
dolo
gy i
s sh
own
to b
e ap
plic
able
for
C-t
erm
inal
seq
uenc
e an
alys
is o
f SD
S-
PA
GE
ele
ctro
blot
ted
prot
eins
as
wel
l.
M6
QU
AN
TIT
AT
IVE
EV
AL
UA
TIO
N O
F D
IFFE
RE
NT
MU
LT
IPL
E A
LIG
NM
EN
T P
RO
GR
AM
S
Hug
h N
icho
las ~'
2 , J
ohn
Hem
pol ~
, Am
y H
inic
h 3 , D
avid
Dee
rfie
ld 1 ,
Jos
eph
Beh
rman
a 1 , A
lex
Rop
elew
ski ~
Pitts
burg
h Su
perc
ompu
tlng
Cen
ter,
2 Dep
artm
ent
of H
uman
Gen
etic
s, a
nd 3
Dep
artm
ent
of M
olec
ular
G
enet
ies/
Bio
chea
rist
ry, U
nive
rsity
of P
ittsb
urgh
, PA
We
have
dev
ised
a m
etri
c fu
r co
mpa
ring
sequ
ence
alig
nmen
ts a
nd w
ritte
n a
prog
ram
for
com
pari
ng
diff
eren
t alig
nmen
ts o
f any
set o
f seq
uenc
es, a
s ge
nera
ted
eith
er f
rom
dif
fere
nt s
cori
ng p
aram
eter
s w
ith a
si
ngle
alig
nmen
t pro
gram
or
from
ent
irel
y di
ffer
ent a
lignm
ent p
rogr
ams.
The
met
ric
is b
ased
on
quan
titat
iou
of th
e di
ffer
ence
s bet
wee
n th
e pa
th g
raph
s of
alig
ned
pair
s of
sequ
ence
s. W
e ap
plie
d th
is
met
ric
to a
lignm
ents
of c
ytoc
hrom
es, a
ldeh
yde
dehy
drog
enas
es, n
ucle
otid
e bi
ndin
g do
mai
ns (
NB
Ds)
, and
vi
ral c
apsi
d pr
otei
ns (
CPs
). T
he a
ppro
ach
enab
les
quan
titat
ive
com
pari
son
of th
e re
sults
fro
m d
iffe
rent
al
ignm
ent s
trat
egie
s. D
iffe
rent
par
amet
ers
have
bee
n us
ed w
ith G
CG
Pile
up, M
SA (1
), an
d G
otoh
(2)
pr
ogra
ms
to g
ener
ate
alig
nmen
ts fo
r co
mpa
riso
n w
ith a
man
ual a
lignm
ent o
f 15
ald
ehyd
e de
hydr
ogen
ase
sequ
ence
s (3
). S
imila
rly,
alig
nmen
ts o
f NB
Ds
and
CPs
wer
e co
mpa
red
to th
ose
gene
rate
d fr
om t
he
supe
rpos
ition
of 3
D c
ryst
al st
ruct
ures
. O
ne s
et o
f (no
n-de
faul
t) p
aram
eter
s w
ith th
e G
otoh
pro
gram
m
ost c
lose
ly re
sem
bled
the
man
ual v
ersi
on, a
nd th
ese
para
met
ers
wer
e us
ed w
ith fa
vora
ble
resu
lts to
m
imic
stru
ctur
e-ba
ased
alig
nmen
ts o
f NB
Ds
and
CPs
. Su
ppor
ted
by N
1H R
R06
009,
NSF
ASC
890
2826
and
a W
m. K
eck
Fdn.
stip
end
to A
H
1. D
J. L
ipm
an e
t al
., Pr
ec. N
atl.
Aca
d. S
ci. U
SA 8
6, 4
412
(198
9).
2. O
. Got
oh, C
AB
IOS
9, 3
61 (1
993)
. 3.
J. H
cmpe
l et
aL, P
rote
in S
cien
ce 2
, 189
0 (19
93).
N1
DEGRADATIVE MODIFICATIONS OF rhlL-ira (RECOMBINANT HUMAN
INTERLEUKIN RECEPTOR ANTAGONIST)
Lori Nixon, Leonard Maneri
Synergen, Inc. , B
oulder, Colorado USA
Recombinant human interleukin-i receptor antagonist (rhlL-ira) is
a protein expressed from E. coli in a soluble, active form.
Several
interesting variants have been characterized, which result from
manufacturing or degradative processes.
These variants include
oxidative modifications, a stable succinimide variant, and a
"cyclized" form of the protein in which the N- and C-termini react.
Analytical techniques used to characterize the variants included
ESl-mass spectrometry (and tandem mass spectrometry), tryptic and
GIu-C peptide maps, IEF, HPLC, SDS-PAGE, bioactivity, etc.
N2
AU
TO
MA
TE
D P
RE
PA
RA
TIO
N O
F C
OM
PL
EX
BIO
LO
GIC
AL
SA
MP
LE
S P
RIO
R T
O
PR
OT
EIN
OR
PE
PT
IDE
SE
QU
EN
CE
AN
AL
YS
IS
Ker
ry N
ug
ent
and
Ken
Sto
ney
, M
ich
rom
Bio
Res
ourc
es,
Au
bu
rn,
CA
956
03
Alt
ho
ug
h m
oder
n an
alyt
ical
tec
hniq
ues
are
very
pow
erfu
l fo
r ch
arac
teri
zati
on o
f pr
otei
ns a
nd p
epti
des
at t
race
lev
els
in c
ompl
ex b
iolo
gica
l sa
mpl
es,
man
y s
amp
les
requ
ire
som
e de
gree
o
f pr
epar
atio
n pr
ior
to f
inal
an
alys
is.
Tec
hniq
ues
such
as
conc
entr
atio
n, d
esal
ting
, b
uff
er e
xch
ang
e an
d de
terg
ent r
emov
al a
re u
sual
ly p
refo
rmed
o
ff f
ine,
whi
ch i
s of
ten
tim
e co
nsu
min
g a
nd c
an r
esul
t in
los
ses
of
the
anal
ytes
of
inte
rest
wh
en w
orki
ng a
t lo
w p
icom
ole
leve
ls.
A n
ew t
echn
ique
has
bee
n de
velo
ped
wh
ich
al
low
s th
e u
se
of
mic
ro
trap
ca
rtri
dges
fo
r au
tom
ated
, on
li
ne
sam
ple
pr
epar
atio
n pr
ior
to f
inal
ana
lysi
s.
Th
ese
trap
car
trid
ges
are
pack
ed w
ith
a va
riet
y o
f pa
ckin
g m
ater
ials
, de
pend
ing
on t
he a
ppli
cati
on o
f in
tere
st,
and
have
bee
n su
cces
sful
ly
empl
oyed
in
a va
riet
y o
f bi
oche
mic
al a
ppli
cati
ons.
A
pro
tein
tra
p ca
rtri
dge
has
bee
n
used
to
conc
entr
ate
and
desa
lt p
ico
mo
le l
evel
s o
f pr
otei
n fr
om
sev
eral
mil
lili
ters
of
0.5
M s
alt
solu
tion
fro
m a
lar
ge s
cale
ion
ex
chan
ge
puri
fica
tion
, w
ith
the
fina
l sa
mp
le
isol
ated
in
a fe
w m
icro
lite
rs o
f vo
lati
le s
olve
nt f
or s
eque
nce
anal
ysis
. A
pep
tide
tra
p ca
rtri
dge
has
been
use
d su
cces
sful
ly w
ith
crud
e sy
nthe
tic
pept
ide
sam
ples
, al
low
ing
conc
entr
atio
n an
d re
mov
al o
f no
nvol
atil
e sa
mpl
e co
mpo
nent
s so
tha
t sa
mpl
es c
an b
e an
alyz
ed
wit
hout
int
erfe
renc
es o
n th
e se
quen
cer.
F
inal
ly,
a va
riet
y o
f de
terg
ent
rem
oval
car
trid
ges
hav
e be
en d
evel
oped
to
allo
w r
emov
al o
f ex
cess
ioni
c an
d no
nion
ic
surf
acta
nt f
rom
pep
tide
and
pro
tein
sam
ples
.
N3
A
NE
W
PT
H
AM
INO
A
CID
S
EP
AR
AT
ION
S
YS
TE
M
TO
IM
PR
OV
E
PE
RF
OR
MA
NC
E A
T T
HE
LO
W P
ICO
MO
LE
LE
VE
L F
OR
OL
DE
R S
EQ
UE
NC
ER
S
Ker
ry N
ug
ent
and
John
Wie
ser,
Mic
hro
m B
ioR
esou
rces
, A
ub
urn
, C
A 9
5603
Co
mm
erci
al p
rote
in s
eque
ncer
s h
ave
bee
n a
vail
able
for
ove
r te
n ye
ars,
and
man
y
labs
con
tinu
e to
get
goo
d re
sult
s at
low
pic
om
ole
lev
els
by c
aref
ully
opt
imiz
ing
and
m
aint
aini
ng th
ese
syst
ems.
Pro
ble
ms
wh
ich
con
tinu
e to
pla
gue
user
s in
clud
e di
ffic
ulty
in
ach
ievi
ng r
epro
duci
ble
tran
sfer
s ba
sed
on t
ime
and
pres
sure
, sh
ifti
ng r
eten
tion
ti
mes
ove
r th
e li
fe o
f th
e P
TH
AA
co
lum
n,
vari
able
co
lum
n l
ife,
re-
opti
miz
atio
n o
f th
e P
TH
AA
se
para
tion
wit
h ea
ch n
ew c
olu
mn
, an
d va
riab
ilit
y in
ach
ievi
ng c
om
ple
te
reso
luti
on o
f al
l am
ino
aci
ds a
nd s
eque
ncer
im
puri
ties
. A
new
sep
arat
ion
syst
em h
as
been
dev
elop
ed t
o el
imin
ate
man
y o
f th
ese
prob
lem
s, a
nd w
orks
wel
l w
ith
exis
tin
g
sequ
ence
r H
PL
C h
ardw
are
wit
h a
min
imu
m o
f m
odif
icat
ion.
T
his
sy
stem
uti
lize
s a
~ 2
.0 x
15
0 m
m 3
u 10
0A R
elia
sil
PT
H
colu
mn
and
a n
ow s
olve
nt b
uff
er c
hem
istr
y
(wit
hout
T
HF
),
and
incl
udes
an
au
tom
ated
co
lum
n
was
h
desi
gned
to
im
pro
ve
repr
oduc
ibil
ity
and
exte
nd c
olu
mn
lif
e.
Wh
en c
oupl
ed w
ith
a pr
otei
n se
quen
cer,
100
ou
t o
f 12
0 ul
of
sam
ple
can
accu
rate
ly a
nd r
epro
duci
bili
ty b
e tr
ansf
erre
d to
the
HP
LC
sy
stem
. D
ata
wil
l be
sho
wn
whi
ch i
nclu
des
base
line
res
olut
ion
of
all
20 P
TH
am
ino
ac
ids
and
the
com
mo
n s
eque
ncer
byp
rodu
cts
(DT
T,
DM
PT
U,
DP
TU
and
DP
U)
in a
to
tal
cycl
e ti
me
of
30 m
inu
tes,
wit
h li
near
ity
of
resp
onse
in
the
1 -
100
pic
om
ole
ra
nge.
S
eque
nce
run
s fr
om
a v
arie
ty o
f pr
otei
ns a
nd p
epti
des
anal
yzed
usi
ng
thi
s n
ew
syst
em o
n se
vera
l ty
pes
of
sequ
ence
rs w
ill
be s
how
n.
N4
> > p=l t J1
e J
I
AR
RE
STI
N T
OP
OG
RA
PH
IC S
TU
DY
US
ING
SE
LEC
TIV
E C
HE
MIC
AL
MO
DIF
ICA
TIO
NS
A
ND
HY
DR
OG
EN
-DE
UTE
RIU
M E
XC
HA
NG
E A
NA
LYZ
ED
BY
MA
SS
SP
EC
TRO
ME
TRY
Hiro
shi O
hgur
o*,
Krz
yszt
of P
alcz
ewsk
i*t,
Ken
neth
A. W
alsh
~ t, R
icha
rd S
. Joh
nson
. ~;
Dep
ts. o
f Oph
thal
mol
ogy*
, P
harm
acol
ogy1
, and
Bio
chem
istry
~ t,
Uni
v. o
f Was
hing
ton,
S
eattl
e, W
ashi
ngto
n 98
195-
0001
, U
.S.A
.
Arr
estin
, a
48 k
iloda
ltons
(kD
a) p
rote
in in
rod
pho
tore
cept
or ce
lls,
is in
volv
ed in
que
nch-
in
g th
e ph
otot
rans
duct
ion
path
way
by
bind
ing
to th
e ph
otoa
ctiv
ated
and
pho
spho
ryla
ted
form
of
rhod
opsi
n (1
). T
o st
udy
dyna
mic
con
form
atio
nal c
hang
es in
arr
estin
, st
ruct
ural
an
alys
is w
as p
erfo
rmed
by
mas
s sp
ectro
met
ry (
MS
) af
ter t
hree
diff
eren
t che
mic
al m
odifi
- ca
tions
; ace
tyla
tion
of ly
sine
res
idue
s, m
odifi
catio
n of
arg
inin
e re
sidu
es b
y 2,
3-bu
tadi
one,
an
d hy
drog
en-d
eute
rium
exc
hang
e. F
ree
arre
stin
and
arr
estin
inc
ubat
ed w
ith p
hosp
hor-
yl
ated
pho
toly
zed
rhod
opsi
n w
as m
odifi
ed a
t di
ffere
nt t
ime
poin
ts,
and
subj
ecte
d to
lim
ited
prot
eoly
sis
follo
wed
by
LC/M
S.
The
kin
etic
s of
the
mod
ifica
tions
at
spec
ific
site
s in
the
arre
stin
mol
ecul
e pr
ovid
ed s
igni
fican
t in
form
atio
n re
gard
ing
dyna
mic
cha
nges
in
prot
ein
stru
ctur
e du
ring
arre
stin
bin
ding
1o
phos
phor
ylat
ed rh
odop
sin.
Thi
s co
mbi
natio
n of
sel
ectiv
e ch
emic
al m
odifi
catio
ns a
nd m
ass
spec
trom
etric
ana
lysi
s m
ay b
ecom
e a
pow
- er
ful s
trate
gy fo
r un
ders
tand
ing
prot
ein
conf
orm
atio
nal
chan
ges
in g
ener
al. S
uppo
rted
by
NIH
Gra
nts
EY
0933
9 ar
id E
Y01
730;
Hum
an F
ront
iers
in S
cien
ce P
rogr
am; a
nd b
y th
e M
ochi
da M
emor
ial F
ound
atio
n fo
r M
edic
al a
nd P
harm
aceu
tical
Res
earc
h. K
P is
a J
ules
an
d D
oris
Ste
in P
rofe
ssor
of R
esea
rch
to P
reve
nt B
lindn
ess,
Inc
.
1.
Hof
man
n et
aL,
J. B
iol.
Che
m.,
267,
157
10-1
5706
(19
92).
Ol
CHARACTERIZATION
OF
ANTIGENIC
DETERMINANTS
OF
P64K
A NEISSERIA
MENINGITIDIS
MEMBRANE PROTEIN.
G.
Padr6n,
V.
Morera,
L.J.
Gonz~lez,
u
T~mbara,
V.
Besada,
R.
Villalonga,
G. Chinea, O. Reyes, H. Garay R.Bringas and C.
Naz~bal.
Center for Genetic Engineering &
Biotechnology,
POB 6162 La Habana Cuba.
We report
the characterization
and determination
of
the most
relevant
epitopes of the protein P64K, an external membrane protein found in most
of the Neisseria meningitidis
strains,that has been considered as a
good
candidate for vaccine development.
P64K
coding
gene
was
cloned
and
expressed
in
E.coli I.
The
protein
sequence
was
determined
by Edman
degradation
and CID-linked
scan mass
spectrometry.
Disulfide bridges and free cysteines were also located.
The probable
antigenic
sites were predicted based on the similarity of
P64K with
a lipoamide
dehydrogenase
from Azotobacter
vinilandii
which
3D
structure
is
known.
Synthetic
and
enzy~natic
peptides
(obtained
by
enzymatic
treatments
of
P64K)
were
tested
by ELISA with
a policlonal
antibody
and
patient
sera.
We
found
a good
correlation
with
the
predicted epitopes
1-Silva.
R et.al. EP 0474 313 A2
(1992).
P2
TH
E U
SE
OF
MA
LD
ITO
F M
AS
S S
PE
CT
RO
ME
TR
Y A
ND
AM
INO
AC
ID
SE
QU
EN
CE
AN
AL
YS
IS I
N C
HA
RA
CT
ER
ISIN
G S
MA
LL
AM
OU
NT
S O
F N
- B
LO
CK
ED
PR
OT
EIN
Leo
nard
C P
ackm
an 1
, Car
l Web
ster
2 an
d Jo
hn G
ray 2
Cam
brid
ge C
entr
e fo
r M
olec
ular
Rec
ogni
tion
, Dep
artm
ents
of
l Bio
chem
istr
y an
d 2p
lant
Sci
ence
s, U
nive
rsit
y of
Cam
brid
ge, T
enni
s C
ourt
Roa
d, C
ambr
idge
, C
B2
1QW
, U
K
One
hun
dred
pic
omol
es o
f a
high
mob
ilit
y gr
oup
(HM
G)
prot
ein
wer
e is
olat
ed b
y re
vers
e ph
ase
hplc
fro
m
an e
xtra
ct o
f pe
a nu
clei
. A
min
o ac
id
sequ
ence
an
d co
mpo
siti
on a
naly
sis
of h
alf
the
sam
ple
show
ed t
he p
rote
in t
o be
blo
cked
at
the
N-
term
inus
. T
he r
emai
ning
50p
mol
was
~ub
ject
ed t
o di
gest
ion
wit
h tr
ypsi
n an
d th
e pe
ptid
es w
ere
sepa
rate
d on
rev
erse
pha
se h
plc.
Eac
h el
uted
pea
k w
as e
xam
ined
by
MA
LD
ITO
F
mas
s sp
ectr
omet
ry a
nd a
min
o ac
id s
eque
nce
anal
ysis
. F
rom
th
e re
sult
ing
info
rmat
ion,
it
was
cle
ar t
hat
the
targ
et p
rote
in s
eque
nce
corr
elat
ed w
ith
the
infe
rred
seq
uenc
e of
a p
revi
ousl
y un
iden
tifi
ed s
egm
ent
of c
lone
d D
NA
. T
here
w
as l
ittl
e po
st-t
rans
lati
onal
mod
ific
atio
n of
the
pro
tein
evi
dent
, bu
t th
e ex
pres
sed
prot
ein
was
sm
alle
r th
an t
he D
NA
seq
uenc
e su
gges
ted.
The
ide
ntit
y of
the
N-
term
inal
blo
ck w
as e
stab
lish
ed a
s w
ell
as t
he s
ite
of C
-ter
min
al p
roce
ssin
g. T
his
wor
ks i
llus
trat
es h
ow e
xten
sive
am
ount
s of
dat
a ca
n be
der
ived
fro
m a
sm
all
amou
nt
of p
rote
in b
y th
e co
mbi
ned
use
of s
eque
nce
anal
ysis
and
M
AL
DIT
OF
m
ass
spec
trom
etry
.
PI
A
ST
RA
IGH
TF
OR
WA
RD
M
ET
HO
D
FO
R
AM
INO
-TE
RM
INA
L
SE
QU
EN
CIN
G
OF
G
AM
MA
-CA
RB
OX
YG
LU
TA
MIC
A
CID
U
SIN
G
ST
AN
DA
RD
ED
MA
N C
HE
MIS
TR
Y.
Bru
ce P
. P
arki
nson
, 1 K
ent A
. Yam
ada2
and
Ann
e R
ando
lph 1
. lC
OR
The
rape
utic
s, In
c., S
outh
San
Fra
ncis
co, C
A 9
4080
and
2pe
rkin
Elm
er,
Fost
er C
ity, C
A 9
4404
. A
pro
cedu
re h
as b
een
deve
lope
d fo
r th
e N
-ter
min
al s
eque
ncin
g of
gam
ma-
ca
rbox
yglu
tam
ic a
cid
(Gla
). T
his
carb
oxyl
ated
am
ino
acid
is
foun
d in
the
K-
depe
nden
t blo
od c
oagu
latio
n pr
otei
ns a
s w
ell a
s se
rum
pro
tein
s C
, S
and
Z a
nd th
e 49
-res
idue
bon
e pr
otei
n, o
steo
cale
in.
Cur
rent
met
hods
for
seq
uenc
ing
Gla
re
sidu
es
(Cai
rns
et a
L,
(199
1)
Ana
lytic
al B
ioch
emis
try
199,
93
-97)
in
volv
e m
ethy
latio
n of
the
sam
ple
prio
r to
sequ
enci
ng t
o re
duce
the
pola
rity
of th
e re
sulti
ng
AT
Z d
eriv
ativ
e an
d th
us i
mpr
ove
extr
actio
n fr
om a
pol
ybre
ne tr
eate
d gl
ass
fiber
fil
ter.
In a
dditi
on,
mod
ified
con
vers
ion
chem
istr
y is
nec
essa
ry to
ens
ure
com
plet
e m
ethy
latio
n of
Asp
, G
lu a
nd G
la r
esid
ues.
In
con
trast
, th
e m
etho
d de
scrib
ed h
ere
uses
sta
ndar
d E
dman
che
mis
try
cycl
es o
n an
App
lied
Bio
syst
ems
476A
pro
tein
se
quen
cer a
nd d
oes
not i
nvol
ve a
ny s
ampl
e pr
e-tre
atm
ent.
Sam
ples
are
app
lied
to
a gl
ass
fiber
filt
er (
with
n
o
poly
bren
e),
PV
DF
m
embr
ane
or
a B
eckm
an
prot
ein/
pept
ide
sam
ple
supp
ort.
Det
ectio
n of
Gla
res
idue
s is
acc
ompl
ishe
d us
ing
a m
odifi
ed H
PLC
sol
vent
A w
hich
yie
lds
a sh
arp
PTH
-Gla
pea
k ju
st a
fter t
he in
ject
ion
front
. P
hosp
hate
(5
mM
) is
add
ed to
sta
ndar
d A
pplie
d B
iosy
stem
s S
olve
nt A
and
th
e pH
is a
djus
ted
to 3
.75
prio
r to
use.
T
he H
PLC
gra
dien
t has
bee
n op
timiz
ed fo
r th
e re
solu
tion
of P
TH-G
la.
P3
r.#3
Q.
CH
AR
AC
TE
RIS
AT
ION
OF
IN
DIV
IDU
AL
N-
AN
D O
-LIN
KE
D G
LY
CO
SY
LA
TIO
N
SIT
ES
US
ING
ED
MA
N D
EG
RA
DA
TIO
N.
An
tho
ny
Pis
ano
, N
ico
le H
. P
acke
r, J
oh
n W
. R
edm
on
d,
Kei
th L
. W
illi
ams
and
An
dre
w
A.
Go
ole
y.
Mac
qu
arie
Un
iver
sity
, S
yd
ney
, N
. S.
W.,
201
9, A
ust
rali
a.
Gly
cosy
late
d
am
ino
ac
ids
can
b
e re
cov
ered
d
uri
ng
th
e n
orm
al
Ed
ma
n
deg
rad
atio
n p
roce
du
re u
sin
g a
so
lid
-ph
ase
des
ign
ed s
equ
enat
or
(Go
ole
y e
t al
., 19
91;
Pis
ano
et
al.,
1993
).
Ho
we
ve
r, i
t w
as u
ncl
ear
exac
tly
ho
w m
uc
h s
ug
ar r
emai
ned
at
tach
ed t
o a
par
ticu
lar
amin
o a
cid
du
rin
g t
he
acid
cle
avag
e o
f th
e N
-ter
min
al a
min
o
acid
fr
om
th
e p
he
ny
lth
ioc
arb
am
yl
(PT
C)-
pep
tid
e an
d
acid
co
nv
ersi
on
o
f th
e an
ilin
oth
iazo
lan
on
e (A
TZ
)-am
ino
aci
d to
th
e p
hen
ylt
hio
hy
dan
toin
(P
TH
)-am
ino
aci
d.
In p
rin
cip
le,
soli
d-p
has
e E
dm
an d
egra
dat
ion
pro
vid
es t
he
idea
l ch
emic
al m
eth
od
fo
r th
e p
uri
fica
tio
n
of
ind
ivid
ual
g
lyco
form
s as
it
is
no
t li
mit
ed
by
th
e p
rob
lem
s as
soci
ated
w
ith
en
do
gly
cosi
das
e sp
ecif
icit
y.
So
lid
-ph
ase
Ed
ma
n
de
gra
da
tio
n
pro
ceed
s n
orm
ally
th
rou
gh
N-
and
O-g
lyco
slat
ed a
min
o a
cid
s (G
oo
ley
et
al.,
1991
),
incl
ud
ing
ex
ten
siv
ely
O-g
lyco
syla
ted
do
ma
ins
(Pis
ano
et
al.,
1993
).
Sin
ce E
dm
an
d
egra
dat
ion
res
ult
s in
th
e se
qu
enti
al r
emo
val
of
amin
o a
cid
s fr
om
th
e N
-ter
min
us,
in
p
rin
cip
le i
t is
po
ssib
le t
o c
olle
ct e
ach
gly
cosy
late
d a
min
o a
cid
in a
gly
cop
epti
de
and
su
bje
ct i
t to
car
bo
hy
dra
te a
nal
ysi
s.
Her
e w
e sh
ow
th
at u
sin
g m
od
el g
tyco
pep
tid
es i
t is
po
ssib
le t
o co
llec
t th
e P
TH
-gly
coam
ino
aci
d fr
om
th
e se
qu
enat
or
and
ch
arac
teri
se
the
oli
go
sacc
har
ide(
s) u
sin
g h
igh
per
form
ance
an
ion
ex
chan
ge
ch
rom
ato
gra
ph
y a
nd
io
n-s
pra
y m
ass
spec
tro
met
ry.
Go
ole
y e
t al
. (1
991)
BB
RC
178
:119
4-12
01.
Pis~
ino
et a
L (1
99
3)G
lyco
bio
log
y 3
:429
-435
.
P4
LO
CA
LIZ
AT
ION
OF
TR
AN
SGL
UT
AM
INA
SE R
EA
CT
IVE
GL
UT
AM
INE
AN
D L
YSI
NE
RE
SID
UE
S IN
AL
ZH
EIM
ER
AM
YL
OID
BA
4 PE
PTID
E:
CR
OSS
-LIN
KIN
G T
O E
XT
RA
CE
LL
UL
AR
MA
TR
IX
PRO
TE
INS
AN
D c
xaM
RE
CE
PTO
R
Lon
e K
. R
asm
usse
n 1, E
sben
S.
Sere
nsen
~, T
orbe
n E
. Pe
ters
en 1,
Jer
gen
Glie
man
n 2 a
nd P
oul
Hel
min
g Je
nsen
~ ~P
rote
in C
hem
istr
y L
abor
ator
y, U
nive
rsity
of
Aar
hus,
Sci
ence
Par
k, D
enm
ark,
='D
epar
tmen
t of
Med
ical
B
ioch
emis
try,
Uni
vers
ity o
f A
arhu
s, D
enm
ark.
The
~-
amyl
oid
pept
ide
(~A
4),
deri
ved
from
a
larg
er
amyl
oid
prec
urso
r pr
otei
n,
is t
he
prin
cipa
l co
mpo
nent
of
seni
le p
laqu
es i
n A
lzhe
imer
's d
isea
se.
~A4
in m
ultim
eric
for
ms
as w
ell
as i
n ag
greg
ates
w
ith g
lyco
prot
eins
has
bee
n fo
und
to b
e co
nstit
uent
s of n
euri
tic p
laqu
es in
Alz
heim
er's
dis
ease
(1)
. It
has
be
en p
ropo
sed
that
tra
nsgl
utam
inas
e is
inv
olve
d in
the
deve
lopm
ent o
f ab
norm
al p
rote
inac
eous
dep
osits
in
neu
rons
(2)
and
is
able
to c
ross
-lin
k ne
uron
al p
rote
ins
(3).
To
asse
ss w
heth
er tr
ansg
luta
min
ase
coul
d ta
ke p
art
in th
e am
yloi
doge
nic
proc
esse
s in
Alz
heim
er's
dis
ease
, we
exam
ined
full-
leng
th (
1-40
) sy
nthe
tic
flA
4 as
a s
ubst
rate
for
tran
sglu
tam
inas
e. H
ere
we
show
that
the
#A4
pept
ide,
con
tain
ing
one
glut
amin
e an
d tw
o ly
sine
re
sidu
es,
is
able
to
form
hom
opol
ymer
s in
a t
rans
glut
amin
ase-
med
iate
d re
actio
n.
Mor
eove
r, t
rans
glut
amin
ase
cata
!yze
d th
e fo
rmat
ion
of h
eter
opol
ymer
s in
rea
ctio
ns o
f B
A4
with
~x2
M
rece
ptor
, a
cons
titue
nt o
f am
yloi
d pl
aque
s (4
), a
nd w
ith e
xtra
cellu
lar
mat
rix
prot
eins
. In
corp
orat
ion
of
site
-spe
cifi
c pr
obes
fo
llow
ed b
y en
zym
atic
dig
estio
n an
d se
quen
cing
of
trac
er-c
onta
inin
g fr
actio
ns
dem
onst
rate
d th
at
both
L
y# 6,
L
ys 2s
an
d G
in 15
in
~A
4 w
ere
susc
eptib
le
to
cros
s-lin
king
by
tr
ansg
luta
min
ase.
(I
) A
. E
. R
oher
et
al.,
J. B
iol.
Che
m.,
268,
307
2 (1
993)
. (2
) D
. J.
Sel
koe
et a
l., P
roc.
Nat
l. A
cad.
Set
. U
SA,
79,
6070
(19
82).
(3
) C
. C
. J.
Mill
er a
nd B
. H
. A
nder
ton,
J.
Neu
roch
em.,
46,
1912
(198
6).
(4)
I. T
ooya
ma
et a
l., M
ol. C
hem
. N
euro
path
ol.,
18,
153
(199
3).
R2
e~
IDE
NT
IFIC
AT
ION
OF
PRO
TE
INS
RE
CO
RD
ED
IN H
UM
AN
2-D
GE
L P
RO
TE
IN D
AT
AB
ASE
S BY
MA
SS
SPEC
TRO
MET
RIC
PE
PTID
E M
APP
ING
Han
ne H
. Ras
mus
sen I
, Ejv
ind M
ortz
2, M
atth
ias M
ann 3
, Pet
er R
oeps
torf
f 2, &
Julio
E. C
elts
1
I Ins
titut
e of M
edic
al B
ioch
emist
ry an
d Dan
ish C
entre
for H
uman
Gen
ome R
esea
rch,
Aar
hus U
nive
rsity
, DK
-800
0 Aar
hus
C. D
enm
ark,
2Dep
artm
ent o
f Mol
ecul
ar B
iolo
gy, O
dens
e Uni
vers
ity, D
K-5
230 O
dens
e M, D
enm
ark a
nd 3
Euro
pean
M
olec
ular
Bio
logy
Labo
rato
ry, 6
900 H
eide
lber
g, G
erm
any.
A c
ompr
ehen
sive
hum
an ke
ratin
ocyt
e tw
o-di
men
sion
al (2
-D) g
el p
rote
in da
taba
se h
as b
een e
stab
lishe
d to
stud
y th
e ex
pres
sion
leve
ls an
d pr
oper
ties o
f the
thou
sand
s of p
rote
ins t
hat o
rche
stra
te va
riou
s ker
atin
ocyt
e fun
ctio
ns
both
in h
ealth
and
dise
ase,
can
cer i
nclu
ded
(l).
To
date
, abo
ut 1
000
of th
e pr
otei
ns re
cord
ed in
the
hum
an
kera
tinoc
yte 2
-D g
el p
rote
in da
taba
se ha
ve b
een i
dent
ified
by
one o
r a c
ombi
natio
n of t
he fo
llow
ing m
etho
ds: i)
co
mig
ratio
n, it
) Was
tem
blot
ting,
iii)
mic
rose
qaen
cing
and
iv) e
xpre
ssio
n of f
ull l
engt
h cD
NA
s.
The
sys
tem
atic
iden
tific
atio
n of
pro
tein
s has
gai
ned
a ne
w d
imen
sion
with
the
adve
nt o
f co
mpu
ter
prog
ram
s for
sear
chin
g pep
tide m
olec
ular
mas
s dat
abas
es w
ith ex
perim
enta
lly ob
tain
ed pe
ptid
e mas
s map
s (2)
. H
ere
we
use
this
appr
oach
to id
entif
y pro
tein
s rec
orde
d in
the
hum
an ke
ratin
ocyt
e 2-D
gel
pro
tein
data
base
(3)
by d
irec
t MA
LD
I MS
anal
ysis
of to
tal p
eptid
e m
ixtu
res o
btai
ned
from
in s
itu d
iges
ts o
f blo
tted
prot
eins
. The
re
sults
cle
arly
dem
onst
rate
that
pep
tide
mas
s map
s can
be
used
for a
rap
id a
nd s
ensi
tive (
fem
to to
ol ra
nge)
pr
otei
n id
entif
icat
ion
allo
win
g fa
st sc
reen
ing
of p
rote
ins r
ecor
ded
in 2
-D g
el p
rote
in d
atab
ases
. The
mas
s sp
ectr
omet
ric
appr
oach
whe
n co
mbi
ned
with
mic
rose
quen
cing
stre
ngth
en th
e id
entif
icat
ion,
and
add
the
po
ssib
ility
for f
ull c
hara
cter
izat
ion o
f pos
t-tra
nsla
tiona
l mod
ific
atio
ns an
d se
quen
ce va
riatio
ns.
I.
Cel
ts, J
.E. e
t al.
Ele
ctro
phor
esis
199
3, 1
4, 1
091-
1198
2.
M
ann,
M. e
t al.
Bio
L M
ass s
pect
rom
. 199
3, 22
, 338
3.
R
asm
usse
n,
H.H
. et a
l. E
lect
roph
ores
is 1
994,
15,
406-
416
DIF
FE
RE
NT
FO
RM
S O
F A
POL
IPO
PRO
TE
IN A
AN
D A
LC
OH
OL
D
EH
YD
RO
GE
NA
SE
S'I
~D
IED
BY
EL
EC
TR
OSP
RA
Y I
,(2/
MS
Stz
ffan
Ren
lun
d,
Hen
rik
Wad
enst
en I
, An
nik
a p
~2
, P
er P
erss
on
I, A
gn
eta
Joh
zn
sso
nl
and
Per
-Ola
f E
dh
md
I P
har
mac
ia B
iote
eh,
S-7
51 8
2 U
pp
sala
, S
wed
en,
Iph
arm
acia
Bio
Sci
enee
Cen
ter,
S
tock
ho
lm,
Sw
eden
, 2
Dep
artm
ent
of
Phy
siol
ogic
al C
hem
istr
y,
Kar
oli
nsk
a In
stit
ute
, S
tock
ho
lm,
Sw
eden
Ele
ctro
spra
y L
C/M
S h
as
in r
ecen
t y
ears
pro
ved
to
be
effi
cien
t fo
r o
bta
tnto
g
stru
ctu
ral
info
rmat
ion
of
pro
tein
s. T
he
tech
niq
ue
is t
mu
aJly
use
d i
n a
n
anal
ytic
al m
ode.
How
ever
, SM
AR
1 TM
S
yst
em,
des
ign
ed f
or m
icro
-pu
rifi
cati
on
of
pept
ades
an
d p
rote
ins,
has
no
w b
een
eq
uip
ped
wit
h a
po
st e
olu
mn
flo
w s
plit
ter,
p
erm
itti
ng
sim
ult
aneo
us
ES
IMS
an
d a
uto
mat
ic p
eak
fi-
aeti
onat
io~
of p
epti
des
, T
wo
gro
up
s of
pro
tein
s w
ere
stu
die
d;
apoU
popr
otei
n A
(a
reco
mb
inan
t, m
uta
nt
form
. AIM
, an
d t
he
nat
ive
pla
sma
vari
anL
AI)
an
d t
hre
e cl
asse
s of
alc
ohol
d
ehy
dro
gen
ase
[fro
m y
easL
gra
m p
osit
ive
bac
teri
a an
d H
elic
ob
aete
r py
lori
]. T
he
pro
tein
s w
e~'e
dig
este
d w
ith
Lys
-C s
peci
fic
end
op
eptl
das
e an
d 1
0-40
0 p
mo
l o
f th
e d
iges
ts w
ere
sep
arat
ed u
sin
g S
MA
RT
Sy
stem
wit
h a
pR
PC
C2
/C1
8 S
C
2.1
/10
co
lum
n.
Th
e fl
ow r
ate
was
25
or
50 i
t/ra
in a
nd
wit
h t
he
po
st c
olu
mn
fl
ow s
plit
ting
at
a ra
tio
of
1:5,
th
e fl
ow to
th
e eI
ectr
osp
ray
in
~
was
5-I
0
pl/
mln
. M
ass
spec
tra
wer
e re
adil
y o
bta
ined
wit
h h
igh
pre
cisi
on
, p
erm
itti
ng
id
enlJ
fiea
tion
of
pro
tein
fra
gm
ents
dif
feri
ng b
y a
sin
gle
amin
o a
cid
resi
du
e. T
he
pep
tid
es c
olle
cted
wer
e u
sed
for
fu
rth
er a
nal
ysi
s b
y m
ean
s of
oth
er t
ech
niq
ues
.
t/i
R1
R3
AU
TO
MA
TE
D T
WO
-DIM
EN
SIO
NA
L E
LE
CT
RO
PH
OR
ES
IS-L
IQU
ID
CH
RO
MA
TO
GR
AP
HY
FO
R T
HE
MIC
RO
PR
EP
AR
AT
IVE
IS
OL
AT
ION
OF
P
RO
TE
INS
Do
nal
d J
. R
ose,
Gla
xo,
Inc,
, R
esea
rch
Tri
ang
le P
ark,
NC
, 2
77
09
Co
nv
enti
on
al t
wo
-dim
ensi
on
al p
oly
acry
lam
ide
gel
elec
tro
ph
ore
sis
(2D
-PA
GE
) h
as b
een
use
d f
or y
ears
for
th
e h
igh
-res
olu
tio
n s
epar
atio
n a
nd
iso
lati
on
of
pro
tein
s fr
om
co
mp
lex
mix
ture
s.
How
ever
, re
lati
ve t
o o
ther
mo
der
n
sep
arat
ion
tec
hn
iqu
es s
uch
as
hig
h p
erfo
rman
ce l
iqu
id c
hro
mat
og
rap
hy
(H
PL
C)
and
cap
illa
ry e
lect
rop
ho
resi
s (C
E),
2D
-PA
GE
is
lab
or-
inte
nsi
ve
du
e to
m
anip
ula
tio
n o
f th
e ge
l b
etw
een
dim
ensi
on
s an
d p
ost
-ru
n,
has
a n
arro
w
dy
nam
ic r
ang
e fo
r sp
ot
det
ecti
on
, an
d c
an s
ho
w p
oo
r re
cov
ery
of
pro
tein
fro
m
the
get.
In
an
att
emp
t to
add
ress
th
ese
def
iden
cies
, co
nti
nu
ou
s sa
mp
le e
luti
on
tu
be
gel
elec
tro
ph
ore
sis
(nat
ive
and
IE
F) h
as b
een
co
up
led
to
rev
erse
-ph
ase
HP
LC
. T
his
tw
o-d
imen
sio
nal
ele
ctro
ph
ore
sis-
liq
uid
ch
rom
ato
gra
ph
y
syst
em
(2D
-EP
/LC
) se
par
ates
pro
tein
s b
y c
har
ge
in t
he
firs
t d
imen
sio
n a
nd
h
yd
rop
ho
bic
ity
in
th
e se
con
d d
imen
sio
n.
Th
e o
rth
og
on
alit
y o
f th
e se
par
atio
n
mec
han
ism
s re
sult
s in
a h
igh
-res
olu
tio
n s
epar
atio
ns.
F
urt
her
mo
re,
sin
ce t
he
seco
nd
dim
ensi
on
use
s a
con
ven
tio
nal
wat
er/a
ceto
nit
rile
/TF
A e
luen
t, t
he
sep
arat
ed c
om
po
nen
ts
can
eas
ily
be
coll
ecte
d fo
r se
qu
enci
ng
or
cou
ple
d t
o
elec
tros
pray
mas
s sp
ectr
om
etry
. T
his
pap
er d
escr
ibes
th
is t
wo
-dim
ensi
on
al
sep
arat
ion
sy
stem
in
clu
din
g t
he
inte
rfac
e u
sed
bet
wee
n t
he
two
dim
ensi
on
s,
anal
ysis
spe
ed,
repr
oduc
ibil
ity,
res
olu
tio
n,
and
sam
ple
rec
ov
ery
an
d
iden
tifi
cati
on
.
R4
IDE
NT
IFIC
AT
ION
OF
AN
OX
IDA
TIV
E M
OD
IFIC
AT
ION
OF
GL
UT
AM
INE
S
YN
TH
ET
AS
E U
SIN
G E
LE
CT
RO
SP
RA
Y L
C/M
S A
ND
ED
MA
N S
EQ
UE
NC
ING
T
EC
HN
IQU
ES
.
Juli
e Sa
haki
an,
Ale
x A
pffe
l, C
had
Mil
ler,
Rod
ney
L.
Lev
ine ~
H
ewle
tt-P
acka
rd C
oand
~L
abor
ator
y of
Bio
chem
istr
y, N
HL
BI,
Bet
hesd
a, M
D
Cov
alen
t m
odif
icat
ion
of p
rote
ins
is a
cen
tral
mec
hani
sm o
f ce
llul
ar r
egul
atio
n.
Loc
atin
g su
ch m
odif
icat
ions
and
ide
ntif
ing
them
can
be
diff
icul
t, ev
en w
hen
the
full
seq
uenc
e of
the
prot
ein
is k
now
n an
d th
e ty
pe o
f m
odif
icat
ion
has
been
est
abli
shed
. G
luta
min
e S
ynth
etas
e, a
45
8 re
sidu
e pr
otei
n kn
own
to b
e su
scep
tibl
e to
met
al-c
atal
yzed
oxi
dati
on w
ith
an a
lter
atio
n of
His
-269
. U
ntil
now
, ho
wev
er,
the
exac
t na
ture
of
the
mod
ific
atio
n w
as n
ot k
now
n.
The
co
mbi
nati
on o
f en
zym
atic
and
che
mic
al d
iges
tion
s, E
dman
Seq
uenc
ing
tech
niqu
es a
nd
Ele
ctro
spra
y L
C/M
S s
how
s ex
tem
ely
usef
ul s
yner
gy fo
r so
lvin
g su
ch p
robl
ems
for
whi
ch
any
one
of th
e te
chni
ques
alo
ne i
s in
suff
icie
nt.
As
an e
xam
ple,
sev
eral
app
roac
hes
for
com
bini
ng th
e te
chni
ques
and
the
ir r
esul
ts w
ill b
e de
mon
stra
ted
in c
hara
cter
izin
g an
oxi
dati
ve
mod
ific
atio
n of
glu
tam
ine
synt
heta
se.
Com
pari
ng p
epti
de m
aps
of p
rote
olyt
ic d
iges
ts o
f na
tive
and
oxi
dize
d gl
utam
ine
synt
heta
se u
sing
an
HP
589
8B E
SM
S a
llow
ed t
he lo
cati
on o
f th
e m
odif
ied
frag
men
t whi
ch w
as c
olle
cted
and
seq
uenc
ed u
sing
an
HP
G10
05A
Pro
tein
S
eque
ncer
to s
how
a m
odif
ied
resi
due
at p
osit
ion
269~
Onc
e lo
cate
d, t
he m
odif
ied
resi
due
coul
d be
iso
late
d us
ing
a si
mpl
e pr
oced
ure
by s
eque
ncin
g a
in-s
itu
CN
Br
dige
st o
f the
ox
idiz
ed g
luta
min
e sy
nthe
tase
pre
pare
d w
ith
the
HP
G10
04B
Pro
tein
Seq
uenc
ing
Stat
ion.
T
he i
sola
ted
PT
H-a
min
o ac
id c
ould
the
n be
fur
ther
cha
ract
eriz
ed u
sing
ES
/MS
. U
sing
this
ap
proa
ch,
the
mod
ifie
d ox
idze
d re
sidu
e ca
n be
ten
tate
vely
iden
tifi
ed a
s 2-
oxo-
His
tidi
ne.
$2
Ma
0r
DE
TE
RM
INA
TIO
N O
F C
YS
TE
INE
BY
AM
INO
AC
ID A
NA
LYS
IS:
ON
-LIN
E O
XID
AT
ION
AN
D
HY
DR
OLY
SIS
Rag
na S
ack
and
Pet
er E
. H
unzi
ker
Bio
chem
isch
es I
nstit
ut,
Uni
vers
it~it
Zi.ir
ich,
Win
tert
hure
rstr
190
, C
H-8
057
Z0r
ich,
Sw
itzer
land
The
det
erm
inat
ion
of c
yste
ine
resi
dues
by
amin
o ac
id a
naly
sis
is s
till
coup
led
with
a n
umbe
r of
pr
oble
ms.
H
owev
er,
its q
uant
ifica
tion
in h
ydro
lysa
tes
is i
mpo
rtan
t fo
r th
e ch
arac
teri
zatio
n of
pr
otei
ns a
nd p
eptid
es.
Qua
ntifi
catio
n of
cys
tein
e re
quire
s ch
emic
al m
odifi
catio
n of
its
fre
e th
iol
grou
p pd
or t
o hy
drol
ysis
and
ana
lysi
s.
The
em
ploy
ed m
etho
ds a
re u
sual
ly t
ime
cons
umin
g, n
ot
alw
ays
quan
titat
ive
and
very
oft
en a
ccom
pani
ed w
ith s
ampl
e lo
sses
. W
e ar
e ve
ry f
requ
ently
enc
ount
ered
with
sam
ples
whe
re t
he a
naly
sis
of t
he c
yste
ine
cont
ent
is
of m
ajor
impo
rtan
ce.
Thu
s, w
e ha
ve a
dapt
ed th
e pe
rfor
mic
aci
d ox
idat
ion
of c
yste
ine
and
cyst
ine
resi
dues
[1]
on
our
amin
o ac
id a
naly
zer
(Mod
el 4
20A
/H;
App
lied
Bio
syst
ems
Inc.
). W
ith t
he n
ew
met
hod
deve
lope
d, s
ampl
es a
re l
oade
d on
to t
he s
ampl
e ca
rtdd
ge o
f th
e an
alyz
er a
nd o
xida
tion
is p
erfo
rmed
aut
omat
ical
ly i
mm
edia
tely
bef
ore
hydr
olys
is.
The
det
ectio
n lim
it of
the
res
ultin
g cy
stei
c ac
id i
s ab
out
10 p
mol
, an
d th
us,
the
sens
itivi
ty o
f cy
stei
ne a
naly
sis
is in
the
sam
e ra
nge
as f
or a
ll ot
her
amin
o ac
ids.
The
ave
rage
err
or o
f ap
prox
imat
ely
9% f
or t
he c
alcu
late
d cy
stei
ne
cont
ent o
f a v
adet
y of
sam
ples
is c
ompa
rabl
e to
that
we
usua
lly o
btai
n on
our
inst
rum
ent f
or o
ther
am
ino
acid
s.
In s
umm
ary,
with
the
aut
omat
ic o
n-lin
e pr
oced
ure
esta
blis
hed
no a
dditi
onal
sam
ple
trea
tmen
t is
ne
cess
ary
for
the
accu
rate
ana
lysi
s of
the
cys
tein
e co
nten
t of
pro
tein
s an
d pe
ptid
es.
[!]
Hirs
C.
H.
W,
, M
etho
ds in
Ezy
mol
., X
I, 1
97 (
1967
)
S1
CH
AR
AC
TER
IZA
TIO
N O
F A
BIN
DIN
G S
ITE
OF
THE
HU
MA
N IM
MU
NO
DEF
ICIE
NC
Y V
IRU
S T
YPE
l R
NA
FO
R T
HE
NU
CLE
OC
APS
ID PR
OTE
IN P
7.
Kaz
uyas
u Sa
kagu
chi,
Nic
ola
Zam
bran
o,
Mar
c S.
L
ewis
1,
Eri
c T
. B
aldw
in 2,
B
ruce
A.
Shap
iro 3
, Jo
hn
W
Eri
ckso
n 2,
Jam
es
G.
Om
ichi
nski
4,
G.
Mar
ius
Clo
re 4,
A
ngel
a M
. G
rone
nbor
n 4,
Eno
re
App
ella
L
ab.
of C
ell
Bio
l. an
d 3L
ab.
of M
ath.
Bio
l.,
NC
I, 1B
EIP
, N
CR
R,
and
2Str
uctu
ral
Bio
chem
., PR
I/D
ynC
orp,
NC
I-FC
RD
C.
4Lab
. of
Che
mic
al P
hysi
cs.,
NID
DK
, U
.S.A
.
The
nu
cleo
caps
id
prot
ein
NC
p7
of H
IV-I
is
im
port
ant
for
enca
psid
atio
n of
th
e vi
ral
geno
me,
R
NA
di
mer
izat
ion,
an
d pr
imer
tR
NA
an
neal
ing
in
vitr
o.
We
have
sho
wn
that
the
psi
reg
ion
of
HIV
-I
RN
A c
an b
e fo
lded
in
two
stem
-loo
p st
ruct
ures
whi
ch a
re r
ecog
nize
d by
an
NC
p7 p
eptid
e (1
).
How
ever
, the
site
s in
the
nuc
leot
ide
sequ
ence
s cr
itica
l fo
r N
Cp7
bin
ding
are
not
kno
wn.
In
th
is
wor
k,
we
have
now
in
vest
igat
ed
the
conf
orm
atio
n of
the
5'
-ter
min
al
regi
on
of
the
HIV
-1
geuo
mic
R
NA
us
ing
the
met
hod
of
Zuc
ker,
and
perf
orm
ed
anal
ytic
al
ultr
acen
trif
ugat
ion
and
chem
ical
in
terf
eren
ce
expe
rim
ents
us
ing
chem
ical
ly
synt
hesi
zed
NC
p7(1
-55)
an
d R
NA
s.
Com
pute
r "p
atte
rn
mat
ch"
anal
ysis
sh
owed
tha
t th
e va
st m
ajor
ity
of p
redi
cted
st
ruct
ures
fo
r th
e ps
i re
gion
(>
80%
) in
clud
ed
both
st
em-l
oop
stru
ctur
es.
The
re
sult
s ob
tain
ed
by
ultr
acen
trif
ugal
an
alys
es r
evea
led
that
one
mol
ecul
e of
NC
p7 p
eptid
e fo
rms
a co
mpl
ex w
ith t
wo
mol
ecul
es o
f R
NA
fr
agm
ents
co
ntai
ning
bo
th
stem
-loo
ps.
Nuc
leot
ides
cr
itica
l fo
r N
Cp7
bin
ding
ha
ve b
een
map
ped
to
a si
ngle
si
te
loca
ted
with
in
the
firs
t lo
op.
A
synt
heti
c 19
-bas
e R
NA
ol
igou
ucle
otid
e co
ntai
ning
th
e fi
rst
stem
-loo
p,
desi
gned
to
in
corp
orat
e th
e nu
cleo
tide
dom
ains
id
entif
ied
as
requ
ired
fo
r N
Cp7
bin
ding
, re
tain
ed
the
abili
ty
to
form
th
e sa
me
2:1
RN
A t
o pe
ptid
e co
mpl
ex.
The
se
resu
lts
sugg
est
that
th
is
19-b
ase
nucl
eotid
e re
pres
ents
a
min
imal
N
Cp7
bi
ndin
g si
te
for
HIV
-1
RN
A
dim
eriz
atio
n.
1.
K.
Saka
guch
i et
aL,
Proc
. Nat
l. A
cad.
Sci
. U
SA, 9
0, 5
219
(199
3).
$3
FU
NC
TIO
NA
L A
ND
PR
OT
EIN
AN
AL
YT
ICA
L S
TU
DIE
S O
N T
HE
MA
JOR
OU
- T
ER
ME
MB
RA
NE
PR
OT
EIN
OF
CA
MP
YL
OB
AC
TE
R J
F.J
UN
I
Wer
ner S
ehrt
der
and
Irm
gard
Mos
er
Frei
e U
nive
rsiti
t Ber
lin, I
nstit
ut fi
ir B
ioeh
emie
und
Insf
itut f
lit M
ikro
biol
ogin
, Fab
ecks
tr. 3
6A, D
-141
95
Ber
lin, F
eder
al R
epub
lic o
f Ger
man
y
Var
ious
mem
bers
of
the
genu
s C
ampy
loba
cter
are
rec
ogni
zed
to c
ause
hum
an d
iarr
hoe
wor
ldw
ide.
T
he m
ajor
out
er
mem
bran
e pr
otei
n (M
OM
P,
42kD
a)
of
C.j
ejun
i w
as
foun
d by
in
vitr
o in
hibi
tion
exp
erim
ents
(E
LIS
A)
to b
e in
volv
ed i
n ad
hesi
on o
f th
e ba
cter
ia t
o eu
eary
otic
cel
l m
embr
anes
(1)
. T
he p
rote
in w
as i
sola
ted
and
puri
fied
by
prep
arat
ive
gel
elee
trop
hore
sis.
Pur
ity
was
che
eked
by 2
D-0
EF
) el
eetr
opho
resi
s. T
he
prot
ein
was
cha
rcte
rize
d by
N-t
erm
inal
seq
uenc
ing
and
amin
o ac
id a
naly
sis.
Pep
tide
s w
ere
gene
rate
d w
ith
diff
eren
t pr
otea
ses
and
CN
Br,
pep
tide
s w
ere
sepe
rate
d by
HP
LC
an
d id
enti
fied
by
sequ
ence
ana
lysi
s. N
o ho
mol
ogy
was
fou
nd i
n da
ta b
ase
to o
ther
m
embr
ane
prot
eins
of
othe
r gr
am-n
egat
ive
bact
eria
. T
he p
rote
in s
how
ed n
o po
rin-
like
he
havi
our
whe
n su
bjec
ted
to S
DS
-PA
GE
. P
reli
min
ary
calc
ulat
ion
of h
ydro
phob
icit
y su
gges
ts t
hat
it i
s no
t a
tran
smem
bran
e pr
otei
n.
Bec
ause
of
its
high
ly i
mm
unog
enic
ch
arac
ter
the
prot
ein
is a
pro
mis
sing
can
dida
te f
or g
ener
atin
g an
eff
icie
nt v
acci
ne.
1.
I.
Mos
er,
W.
Sch
r6de
r,
and
E.
Hel
lman
n, M
ed.
Mic
robi
ol.
Imm
unoL
Vol
.180
pp
289-
303
S4
RE
LA
XA
SE
(T
RA
I) O
F I
neP
ot
PL
AS
MID
RP
4 C
AT
AL
YZ
ES
A S
ITE
- S
PE
CIF
IC C
LE
AV
ING
-JO
ININ
G R
EA
CT
ION
OF
SIN
GL
E-S
TR
AN
DE
D D
NA
Wcr
ner S
chrS
der t,
Wer
ner P
anse
grau
2, a
nd E
rich
Lan
ka 2
1Fre
ie U
nive
rsit~
t Ber
lin, I
nstit
at fi
ir B
ioch
emie
, Fab
ecks
tras
se 36
A, D
-141
95 B
erlin
, Fed
eral
R
epub
lic o
f Ger
man
y; an
d 2M
ax-P
lane
k-ln
stite
t fiir
Mol
ekul
are G
enet
ik, A
btei
lung
Sch
uste
r, Il
mes
tras
se 73
, D-1
4195
Ber
lin, F
eder
al R
epub
lic of
Ger
man
y
Con
juga
tive
DN
A t
rans
fer
of t
he s
elf-
tran
smis
sibl
e bro
ad-h
ost-
rang
e pl
asm
id R
P4
is
init
iate
d by
str
and-
and
sit
e-sp
ecif
ic c
leav
age
at t
he n
ick
site
(ni
c) o
f th
e tr
ansf
er
orig
in (
or/T
). C
leav
age
resu
lts
in c
oval
ent a
ttac
hmen
t of
the
pla
smid
-enc
oded
rel
axas
e (T
raI)
to
the
5" -t
erm
inal
2"
-deo
xycy
tidi
ne r
esid
ue a
t ni
c. W
e de
mon
stra
te th
at T
yr 22
is
the
cen
ter
of t
he c
atal
ytic
sit
e of
Tra
I, m
edia
ting
cle
avag
e vi
a fo
rmat
ion
of a
ph
osph
odie
ster
bet
wee
n D
NA
5 "
phos
phor
yl a
nd t
he a
rom
atic
hyd
roxy
l gr
oup.
The
sp
ecif
icit
y of
cle
avag
e se
en w
ith
form
I
oriT
DN
A w
as v
erif
ied
wit
h sh
ort
olig
odeo
xyri
bonu
cleo
tide
s em
brac
ing
the
nick
reg
ion.
The
rea
ctio
ns d
escr
ibed
her
e fu
rthe
r su
ppor
t th
e hy
poth
esis
that
DN
A t
rans
fer
via
conj
ugat
ion
invo
lves
a r
olli
ng
circ
le-l
ike
mec
hani
sm w
hich
gen
erat
es t
he i
mm
igra
nt s
ingl
e st
rand
whi
le D
NA
-bou
nd
Tra
I pr
otei
n sc
ans
for
the
occu
rren
ce o
f a
seco
nd c
leav
age
site
at
the
dono
r-re
cipi
ent
inte
rfac
e.
1.)
W.
Pan
segr
au e
t al
., P
roc.
Nat
. A
cad.
Sci
ence
US
A V
ol.
90 p
p292
5-29
(199
3)
2.)
W.
Pan
segr
au e
t al
., J
ourn
al o
f B
iolo
gica
l C
hem
istr
y, V
ol.
269,
N~
pp
278
2-
2~78
9(19
94)
$5
HIG
H-S
PE
ED
C
HR
OM
AT
OG
RA
PH
IC
SE
PA
RA
TIO
N
OF
P
RO
TE
INS
A
ND
PE
PTID
ES:
A
PP
LIC
AT
ION
T
O
RA
PID
P
EP
TID
E
MA
PP
ING
O
F
IN-G
EL
D
IGE
ST
ED
P
RO
TE
INS
. R
icha
rd J
. Si
mps
on, J
arae
s E
ddes
, H
ong
Ji,
Gav
in E
. R
eid
and
Rob
ert
L.
Mor
itz.
Jo
int
Prot
ein
Stru
ctur
e L
abor
ator
y, L
udw
ig I
nstit
ute
for C
ance
r Res
earc
h an
d T
he W
alte
r and
Eliz
a H
all I
nsti
tute
of
Med
ical
Res
earc
h, P
O R
oyal
Mel
bour
ne H
ospi
tal,
Vic
305
0, A
ustr
alia
.
A n
ew l
iqui
d ch
rom
atog
raph
ic t
echn
ique
has
bee
n de
velo
ped
for
the
high
-spe
ed s
epar
atio
n of
low
m
icro
gram
am
ount
s of
pro
tein
and
pep
fide
. T
his
tech
niqu
e ut
ilise
s co
nven
tiona
l 30
0,~
pore
, 7-
ttm
di
amet
er s
ilica
-bas
ed p
acki
ngs,
gra
dien
t el
utio
n an
d si
gnif
ican
tly h
ighe
r lin
ear
flow
vel
ociti
es.
Thi
s de
crea
ses
the
sepa
ratio
n ti
me,
for
a t
ypic
al m
icro
bore
2.1
mm
ID
car
trid
ge,
by a
lmos
t an
ord
er o
f m
agni
tude
. L
ittle
dif
fere
nce
in re
solu
tion
was
obs
erve
d w
ith th
is h
igh
spee
d ch
rom
atog
raph
ic a
ppro
ach
com
pare
d to
con
vent
iona
l pep
tide
map
ping
pro
cedu
res.
In
add
ition
, w
e ha
ve im
prov
ed o
ur m
etho
d [1
] fo
r ob
tain
ing
pept
ides
fr
om l
ow
mic
rogr
am
amou
nts
of i
n-ge
l di
gest
ed
prot
eins
fo
r st
ruct
ural
ch
arac
teri
zatio
n by
pep
tide
mas
s fi
nger
prin
ting
(util
isin
g ca
pilla
ry c
olun
m L
C/E
SI-m
ass
spec
trom
etry
) an
d E
dman
deg
rada
tion.
T
he l
ong
acid
ext
ract
ion
Step
s hav
e be
en r
epla
ced
by e
xtra
ctin
g fo
r 30
min
w
ith
1%
aq.
TFA
fo
llow
ed
by 0
.1%
T
FA/6
0%
acet
onitr
ile
and
a re
duct
ion
of d
eter
gent
fo
r co
mpa
tibili
ty w
ith E
SI-M
S.
The
gen
eral
util
ity o
f the
se p
roce
dure
s ha
s be
en d
emon
stra
ted
in th
e ra
pid
iden
tific
atio
n of
a w
ide
vari
ety
of 2
D-g
el r
esol
ved
prot
eins
fro
m h
uman
colo
nic
carc
inom
a ce
lls.
1. W
ard,
L.D
.. R
eid.
O.E
., M
oritz
, R
.L.
and
Sim
pson
, R.J
. (19
90)
in C
urre
nt R
esea
rch
in P
rote
in C
hem
istry
: Te
chni
ques
, St
ruct
ure.
an
d Fu
nctio
n. (
Vill
afra
nea,
J.
J. F
,d.)
Aca
dem
ic P
ress
, pp
179-
190.
$6
IDE
NT
IFIC
AT
ION
OF
POST
TR
AN
SLA
TIO
NA
L M
OD
IFIC
AT
ION
S IN
BO
VIN
E O
STE
OPO
NT
IN:
LO
CA
LIZ
AT
ION
OF
28 P
HO
SPH
OR
YL
AT
ION
SIT
ES,
3
O-G
LY
CO
SYL
AT
ION
SIT
ES
AN
D 2
T
RA
NSG
LU
TA
M1N
ASE
RE
AC
TIV
E G
LU
TA
MIN
ES
Esb
en S
. So
rens
en t,
Lon
e K
. R
asm
usse
n ~, P
oul
Hen
ning
Jens
en :,
Pete
r H
ojru
p 3 an
d T
orbe
n E
. Pe
ters
en 1
IPro
tein
Che
mis
try
Lab
orat
ory,
Uni
vers
ity
of A
arhu
s, S
cien
ce P
ark,
Den
mar
k, F
ax:
(45)
861
3656
97
2Dep
artm
ent
of M
edic
al
Bio
chem
istr
y, U
nive
rsit
y of
Aar
hus,
Den
mar
k, 3
Dep
artm
ent
Of
Mol
ecul
ar
Bio
logy
, U
nive
rsit
y of
Ode
nse,
Den
mar
k.
Ost
eopo
ntin
is a
hig
hly
phos
phor
ylat
ed n
on-c
olla
geno
us bo
ne m
atri
x pr
otei
n co
ntai
ning
the
Arg
-Gly
-Asp
- Se
r se
quen
ce re
spon
sibl
e fo
r ce
ll ad
hesi
on. B
one
oste
opon
tin
bind
s to
hydr
oxya
pati
te an
d is
an
impo
rtan
t co
mpo
nent
in th
e ca
lcif
icat
ion
proc
ess.
Ost
eopo
ntin
is a
lso
expr
esse
d in
act
ivat
ed T
-cel
ls a
nd m
etas
tazi
ng
tum
or c
ells
whe
re r
oles
in
the
earl
y re
sist
ance
to
bact
eria
l in
fect
ions
and
bin
ding
of
tum
or c
ells
at
seco
ndar
y si
tes,
res
pect
ivel
y, h
ave
been
hyp
othe
size
d (1
,2).
We
have
isol
ated
ost
eopo
ntin
fro
m b
ovin
e m
ilk
(3)
and
char
acte
rize
d po
sttr
ansl
atio
nal
mod
ific
atio
ns
in t
he p
rote
in.
By
a co
mbi
nati
on o
f m
ass
spec
trom
etry
and
am
ino
acid
seq
uenc
ing o
f eth
anet
hiol
-tre
ated
pep
tides
(4)
, a
tota
l of 2
8 ph
osph
oryl
atio
n si
tes
wer
e lo
caliz
ed.
Furt
herm
ore,
thr
ee O
-gly
cosy
latio
n si
tes
wer
e id
entif
ied.
To
inve
stig
ate
whe
ther
is
opep
tide
bond
ing
coul
d be
inv
olve
d in
the
fun
ctio
ns o
f os
teop
ontin
we
exam
ined
ost
eopo
ntin
as
a su
bstr
ate
for
tran
sghi
tnm
inas
e. I
ncor
pora
tion
of a
rad
iola
belle
d si
te-s
peci
fic
prob
e sh
owed
tha
t tw
o sp
ecif
ic g
lnta
min
es c
ould
act
as
tran
sgln
tnm
inas
e acc
epto
r si
tes.
(1)
V.
Fet
et a
l.,
Gen
omic
s, 5
, 37
5 (1
989)
. (2
) A
.M.
Cra
ig e
t al
., In
t. J.
Can
cer,
46,
133
(19
90).
(3)
E.S
. So
rens
an a
nd T
.E.
Pete
rsen
, J.
Dai
ry R
es.,
60,
189
(199
3).
(4)
H.E
. M
eyer
et
al.,
FEB
S L
ea.,
204
, 61
(19
86).
$7
m,l tim
INT
EG
RA
TIO
N O
F M
AL
DI
MA
SS S
PEC
TR
OM
ET
RY
WIT
H I
N S
ITU
PR
OT
EA
SE D
IGE
STIO
NS
ON
H
IGH
RE
TE
NT
ION
PV
DF
ME
MB
RA
NE
S T
O O
BT
AIN
IN
TE
RN
AL
SE
QU
EN
CE
S A
T M
AX
IMU
M
SEN
SIT
IVIT
Y
Dav
id W
. Sp
eich
er,
Dav
id F
. R
eim
and
Kay
e D
. Sp
eich
er
The
Wis
tar
Inst
itute
, Ph
ilade
lphi
a, P
A 1
9104
, USA
Mat
rix
assi
sted
lase
r de
sorp
tion/
ioni
zatio
n T
OF
mas
s sp
ectr
omet
ry is
a c
ritic
al c
ompl
emen
tary
met
hod
to
conv
entio
nal p
eptid
e se
quen
ce a
naly
sis.
T
he a
mou
nt a
nd r
elia
bilit
y of
seq
uenc
e in
form
atio
n ob
tain
ed i
n th
e lo
w p
icom
ole
to m
id-f
emto
mol
e ra
nge
can
be s
ubst
antia
lly in
crea
sed
by c
ombi
ning
mas
s an
alys
is w
ith
both
in s
itu
prot
ease
dig
estio
ns o
n PV
DF
mem
bran
es a
nd h
igh
perf
orm
ance
seq
uenc
e an
alys
is o
n a
biph
~sic
co
lum
n se
quen
cer.
B
enef
its o
f m
ass
anal
ysis
pri
or t
o se
quen
cing
inc
lude
: de
tect
ion
of m
ost
mix
ture
s,;
dete
ctio
n of
the
long
est p
eptid
es,
the
abili
ty to
set
a p
reci
se n
umbe
r of
seq
uenc
er c
ycle
s, c
onfi
rmat
ion
of
tent
ativ
e se
quen
ce a
ssig
nmen
ts, a
nd d
etec
tion
of p
oten
tial p
osttr
ansl
atio
nal
mod
ific
atio
ns.
To
real
ize
the
last
two
bene
fits
, the
com
plet
e pep
tide
sequ
ence
mus
t be
obta
ined
. R
ecen
tly re
leas
ed v
ersi
on 2
.2 c
hem
istr
y an
d pr
ogra
ms
for t
he H
P G
1005
A b
ipha
sic
cart
ridg
e se
quen
cer p
rovi
de v
ery
high
rep
etiti
ve y
ield
s on
mos
t pe
ptid
es, t
ypic
ally
> 9
4% f
or m
ost p
eptid
es.
Hen
ce m
ost p
eptid
es c
an b
e se
quen
ced
to th
e te
rmin
al re
sidu
e ev
en w
hen
initi
al y
ield
s ar
e in
the
0.5
to 1
0 pm
ole
rang
e.
The
mul
tiple
seq
uenc
er c
olum
n fe
atur
e co
uple
d w
ith p
rede
term
ined
mas
ses
perm
its a
max
imum
sam
ple
thro
ughp
ut b
y au
tom
ated
tand
em a
naly
sis
of th
e co
mpl
ete
pept
ide
sequ
ence
s w
ithou
t per
form
ing
unne
cess
ary
extr
a cy
cles
. B
y op
timiz
ing
elec
trot
rans
fer,
di
gest
ion,
and
han
dlin
g yi
elds
, it i
s fe
asib
le to
rou
tinel
y ob
tain
inte
rnal
seq
uenc
es o
n ex
peri
men
tal s
ampl
es
whe
n as
littl
e as
20
pmol
es a
re l
oade
d on
to a
gel
, w
hich
res
ults
in
a re
cove
ry o
f bet
wee
n 12
to 1
5 pm
oles
on
hig
h re
tent
ion
PVD
F m
embr
anes
. W
ith 1
5 pm
oles
of a
sin
gle
prot
ein
on th
e bl
ot,
reco
veri
es o
f pur
ifie
d fr
actio
ns a
re t
ypic
ally
in
the
10%
to
30%
ra
nge
(1.5
to
4.5
pmol
es/f
ract
ion)
, w
hich
cor
resp
ond
to
conc
entr
atio
ns in
the
low
fem
tom
ole/
#l r
ange
, w
hich
is
com
patib
le w
ith d
irec
t m
ass
mea
sure
men
ts u
sing
M
AL
DI m
ass_
spec
trom
etry
. $8
CH
AR
AC
TE
RIZ
AT
ION
O
F
PU
RIF
IED
Y
EA
ST
P
RO
TE
IN
GE
RA
NY
LG
ER
AN
YL
TR
AN
S F
ER
AS
E
TY
PE
I
US
ING
A
C
ON
TIN
UO
US
F
LU
OR
ES
CE
NC
E
AS
SA
Y.
Wil
liam
G
. St
irta
n an
d C
. D
ale
Poul
ter,
D
epar
tmen
t o
f C
hem
istr
y, U
nive
rsit
y o
f U
tah,
Sal
t L
ake
City
, U
tah
8411
2
Post
tran
slat
iona
l pr
otei
n pr
enyl
atio
n co
nfer
s un
ique
lip
ophi
lic
prop
erti
es t
o se
vera
l ce
llul
ar
prot
eins
an
d is
ne
cess
ary
for
thei
r pr
oper
bi
olog
ical
fu
ncti
on.
Pro
tein
ge
rany
lger
anyl
tran
sfer
ase
type
I (
GG
Tas
e-I)
tra
nsfe
rs a
Cao
ger
anyl
gera
nyl
moi
ety
from
ge
rany
lger
anyl
dip
hosp
hate
, vi
a a
thio
ethe
r lin
kage
, to
a c
yste
ine
resi
due
four
am
ino
acid
s fr
om
the
C-t
erm
inus
o
f ca
ndid
ate
pept
ides
an
d pr
otei
ns.
GG
Tas
e-I
activ
ity
in
Sa
cch
aro
myc
es c
erev
isia
e is
med
iate
d by
a h
eter
odim
eric
enz
yme
enco
ded
by t
he g
enes
R
AM
2 an
d C
DC
43.
Rec
ombi
nant
GG
Tas
e-I
has
been
ove
rexp
ress
ed i
n E
. co
li u
sing
a
mul
tico
py p
lasm
id i
n w
hich
the
RA
M2
and
CD
C43
str
uctu
ral
gene
s w
ere
tran
slat
iona
lly
coup
led
by o
verl
appi
ng t
he T
GA
TG
sto
p-st
art
codo
ns a
nd b
y lo
cati
ng a
rib
osom
e-bi
ndin
g si
te w
ithi
n th
e up
stre
am g
ene.
T
he e
nzym
e w
as p
urif
ied
to a
ppar
ent
hom
ogen
eity
in
two
step
s us
ing
ion-
exch
ange
and
im
mun
oaff
init
y ch
rom
atog
raph
y, th
e la
tter
of
whi
ch w
as m
ade
poss
ible
by
inco
rpor
atin
g a
C-t
erm
inal
tri
pept
ide
(Glu
-Glu
-Phe
) ep
itop
e in
the
ram
2 su
buni
t.
GG
Tas
e-I
effi
cien
tly
mod
ifie
s th
e da
nsyl
ated
pe
ntap
epti
de
Dan
syl-
GC
IIL
whi
ch,
upon
ge
rany
lger
anyl
atio
n, u
nder
goes
a l
arge
flu
ores
cenc
e en
hanc
emen
t an
d al
so e
xhib
its
a sh
ift
in f
luor
esce
nce
emis
sion
. A
con
tinu
ous
fluo
resc
ence
ass
ay h
as b
een
deve
lope
d to
mon
itor
G
GT
ase-
I ac
tivity
an
d to
fur
ther
cha
ract
eriz
e th
is p
rote
in p
reny
ltra
nsfe
rase
.
$10
A T
HIO
L M
ED
IAT
ED
AU
TO
LY
TIC
CLE
AV
AG
E O
F H
OM
OC
YS
TE
INY
L P
RO
LYL
BO
ND
B.R
. S
rini
vasa
and
S.P
. B
arde
A
stra
R
esea
rch
Cen
tre
Indi
a,
P.B
. B
anga
lore
56
0 0
03
, IN
DIA
. N
o.
359,
1
8th
C
ross
M
alle
swa
ram
,
A
syn
the
tic
olig
op
ep
tid
e S
G P
S N
h P
P E
I (h
=
ho
mo
cyst
ein
e)
wa
s fo
un
d
to b
e un
stab
le in
so
luti
on
at
pH b
elo
w 4
. W
hile
pu
rify
ing
thi
s p
ep
tid
e,
it w
as
notic
ed t
ha
t at
am
bie
nt
tem
pe
ratu
re (
25~
th
is p
ep
tid
e e
lute
d as
a b
roa
d
peak
on
reve
rse
phas
e co
lum
n a
nd a
s th
e c
olu
mn
te
mp
era
ture
wa
s ra
ised
to
45
~
the
peak
sha
rpen
ed.
Th
e p
urif
ied
pe
pti
de
in s
olu
tio
n a
t or
be
low
pH
4
wa
s fo
und
to d
eg
rad
e a
nd t
he
pro
du
cts
we
re c
ha
ract
eri
zed
. T
he r
esul
ts
sugg
este
d th
at
the
pe
pti
de
un
de
rgo
es
cle
ava
ge
be
twe
en
th
e h
om
ocy
ste
inyl
pr
olyl
pe
pti
de
bon
d un
der
aci
dic
co
nd
itio
ns
(be
low
pH
4)
to g
ive
pe
pti
de
s S
G
P
S
N
h*
(h*
= cy
cliz
ed
h
om
ocy
ste
ine
) an
d P
P
E
I.
A
rea
ctio
n
mec
hani
sm h
as b
een
pro
po
sed
wh
ich
su
gg
est
s th
at
unde
r a
cid
ic c
on
dit
ion
s th
e im
ide
nitr
ogen
of
prol
ine
ge
ts p
roto
na
ted
and
th
e s
ulfu
r o
f h
om
ocy
ste
ine
th
iol
reac
ts w
ith
th
e c
arb
on
yl o
f h
om
ocy
ste
ine
. T
his
res
ults
in
the
cle
ava
ge
o
f th
e p
ep
tid
e im
ide
bond
to
giv
e a
th
erm
od
yna
mic
ally
sta
ble
five
me
mb
ere
d
pe
pti
dyl
ho
mo
cyst
ein
ly t
hio
lact
on
e a
nd a
te
tra
pe
pti
de
P P
E I.
H
ow
eve
r th
e
dim
eriz
ed p
ep
tid
e o
r a
mo
no
me
r w
ith
a p
rote
cte
d s
ulf
hyd
ryl
is q
uit
e s
tab
le
unde
r ac
idic
co
nd
itio
ns.
$9
THE
DNA-TOPO I SOMERASE
I~
ANALYSIS
BY
LIMITED
P~!OTEOLYSIS
Or
DOMAIN STRUCTURE
AND CONFGRMATIONAL
CHANGES.
Alyona Sukhanova,
Serge'/ Vor.ob"ev, Alexar~der- Gabibov,
Igor. Br.onst ein
Engelhardt
Institute of Molecular- Biology,
Moscow,
Russia
Monoclonal
antibodies
against
to~,o I wer'e generated
and
used to
study structure-function
r.elationshi~,s.
These antibodies
served
to ma~, endog, e~,tidase
cleavage and
e~,ito~,e sites on
to~,o I
and
demonstr.ate that N-terminus
is essential
for- the interaction with
DNA-binding
domain or at least causes chan(les in the ~,roteoiysis
oattern
in this region. The lack of N-terminal
domain is associ-
ated
wit
h
mo
ve
me
nts
o
f th
e
do
ma
ins
in
th
e
en
zy
me
: a
s
a
co
r, s
e-
,~u
en
se
so
me
sit
es
in
to
~o
I
be
co
me
m
ore
s
en
sit
ive
to
c
lea
va
ge
b
y
try
ps
in.
Th
e
pre
se
nc
e
of
DN
A
als
o
ca
us
es
o
ro
min
en
t c
ha
ng
es
in
th
e
pro
teo
lys
is
pa
tte
rn
o
f th
e
en
zy
me
. O
ur.
s
pe
cu
lati
on
is
th
at
ca
sc
ad
ing
o
f a
llo
ste
ric
c
ha
ng
es
a
cro
ss
a
dja
ce
nt
do
ma
ins
pro
vid
es
a
w
ay
of
co
mm
un
ica
tio
n
be
twe
en
c
ata
lyti
c
ce
nte
r,
DN
A-b
ind
in~
re
gio
n
an
d
oth
er
fun
cti
on
al
do
ma
ins
.
$11
=r
PR
EP
AR
AT
ION
AN
D C
HA
RA
CT
ER
IZA
TIO
N O
F A
CO
NJU
GA
TE
OF
AN
NE
X1
N V
A
ND
TH
E B
-CH
AIN
OF
UR
OK
INA
SE
K
enji
Tan
aka,
t "
' 1
. 1
. 2
.. 3
Ku
mk
o E
mag
a,
Min
oru
Tsu
kada
, Jo
nath
an F
. T
art,
K
azu
o F
ujl
kaw
a 1
Res
earc
h D
ivis
ion,
The
Gre
en C
ross
Co
, O
sak
a Ja
pan,
D
epts
. o
f L
abo
rato
ry M
edic
ine
2 an
d
Bio
chem
istr
y, 3
Uni
vers
ity
of
Was
hing
ton,
Sea
ttle
Was
hing
ton.
A
con
juga
te o
f an
nexi
n V
and
the
B-c
hain
of
urok
inas
e w
as p
repa
red
to s
tudy
if
it h
as
an e
nhan
ced
in v
ivo
fibr
inol
ytic
act
ivit
y A
nnex
in V
, w
hich
has
a s
tron
g C
a ++
depe
nden
t-
bind
ing
affi
nity
to n
egat
ivel
y ch
arge
d ph
osph
olip
ids
(1)
or a
ctiv
ated
pla
tele
ts (
2),
is u
sed
for
ta
rget
ing
plat
elet
-con
tain
ing
thro
mbi
. A
n in
terc
hain
dis
ulfi
de b
on
d o
f ur
okin
ase
was
red
uce
d
for
2 hr
s in
lm
M D
TT
and
10
mM
ben
zam
idin
e at
pH
8.0
, an
d th
e B
-cha
in w
as i
sola
ted
usin
g a
benz
amid
ine-
agar
ose
colu
mn.
An
anne
xin
V m
utan
t w
hich
has
an
N-t
erm
inal
ext
ensi
on w
ith
six
amin
o ac
ids
(A-C
-D-H
-S-M
), w
as p
repa
red
by
exp
ress
ing
in E
. co
ll.
Ann
exin
V/B
-ch
ain
co
njug
ate
was
for
med
by
mix
ing
thes
e tw
o c
ompo
nent
s fo
r 20
hrs
at
37~
in
the
pre
senc
e o
f 20
mM
ben
zam
idin
e (a
ir o
xida
tion
) an
d is
olat
ed b
y tw
o a
ffin
ity
colu
mns
, li
poso
me-
and
be
nzam
idin
e-S
epha
rose
. T
he c
onju
gate
mig
rate
d as
a s
ingl
e ba
nd o
f 69
kD
a o
n a
no
n-r
edu
ced
ge
l an
d it
gav
e tw
o b
ands
, th
e m
utan
t an
nexi
n V
(35
kD
a) a
nd t
he B
-cha
in (
30 k
Da)
, on
a
red
uce
d g
el.
Pre
lim
inar
y re
sult
s sh
owed
tha
t th
e co
njug
ate
has
four
fold
gre
ater
fib
rino
lyti
c ac
tivi
ty t
han
urok
inas
e in
a r
at p
ulm
onar
y em
boli
sm m
odel
. T
he c
onju
gate
als
o ca
use
d l
ess
syst
emic
act
ivat
ion
of
fibr
inol
ysis
than
uro
kina
se a
s ju
dg
ed b
y pl
asm
a le
vel
of~
x2-p
lasm
in
inhi
bito
r.
1. T
ait
et a
l.,
J. B
iol.
Che
m.
264,
794
4-79
49 (
1989
).
2. T
hiag
araj
an a
nd T
ait,
J.
Bio
l. C
hem
. 26
5, 1
7420
-174
23 (
1990
).
T1
A N
OV
EL
C-T
ER
MIN
AL
SE
QU
EN
CIN
G M
ET
HO
D U
SIN
G P
ER
FLU
OR
OA
CY
L A
NH
YD
RID
ES
Aki
ra T
sugi
ta,
Mas
aham
Kam
o, K
eiji
Tak
amot
o an
d K
azuo
Sat
eke
Res
earc
h Ins
titut
e fo
r Bin
scin
nces
, Sc
ienc
e U
nive
rsity
of T
okyo
, Yam
azak
i 266
9, N
oda,
Chi
ba 2
78, J
apan
Pep
tide
s ex
pose
d to
the
vapo
r of p
enta
fluo
mpr
opio
nic a
nhyd
ride
pro
vide
the
succ
essi
vely
cle
avag
e of
the
pept
ides
fr
om th
e C
-ter
min
i. T
he p
rodu
cts
wer
e an
alyz
ed b
y FA
B m
ass
spee
trom
effy
(l,2
,3).
Thi
s re
acti
on h
as t
o be
car
ded
out
unde
r sl
rict
wat
er-f
ree
cond
itio
ns, b
ecau
se w
ater
reac
ts w
ith
the
anhy
drid
e an
d fo
rms
the
corr
espo
ndin
g ac
id
inst
antly
, T
he re
acti
on w
as p
ract
ical
ly ca
rrie
d ou
t wit
h ac
id a
nhyd
ride
aeet
onilr
ile s
olut
ion.
How
ever
acet
onitr
ile w
as
foun
d to
be
unes
sent
ial
for t
he re
acti
on to
pro
ceed
, si
nce
a va
por f
rom
the
anhy
drid
e in
the
abse
nce o
f ace
toni
trile
was
ob
serv
ed to
be
still
rea
ctiv
e.
Whe
n w
ater
was
add
ed to
this
rea
gent
(10
:l(r
eol/
mol
)), t
he r
eact
ion
was
int
erru
pted
. T
he a
ddit
ion
of a
ceto
nitr
ile i
n th
is r
eage
nt m
ixtu
re re
acti
vate
the
reac
tion.
In
prac
tice
we
used
a v
acuu
m h
ydro
lysi
s tu
be a
s th
e re
acti
on v
esse
l an
d th
e m
ost o
pera
tion
s w
ere
carr
ied
out i
n a
glov
e bo
x co
ntin
uous
ly fl
ushe
d w
ith
dry
nitr
ogen
gas
to r
id o
f moi
stur
e.
The
deg
rada
tion
reac
tion
acc
ompa
nies
acy
lati
on re
acti
on, w
hich
is s
low
er th
an th
e de
grad
atio
n re
acti
on a
t -20
~ E
xpos
ure
the
reac
tion
pro
duct
to th
e 10
%(v
/v)
pyri
dine
wat
er v
apor
at
100~
fo
r 30
ra
in w
as u
sual
ly e
mpl
oyed
to s
impl
ify
the
spec
trum
. T
his
wat
er v
apor
trea
tmen
t elim
inat
es t
he O
-acy
lati
on p
eaks
(o
xazo
lone
rin
g an
d Se
r) a
s w
ell a
s a
part
of -
18
peak
whi
ch is
due
to
the
oxaz
olon
e for
mat
ion
at th
e C
-ter
min
i of
the
resp
ecti
ve fr
agm
ents
. Mos
t of
the
degr
aded
mol
ecul
ar io
ns a
ccom
pani
ed w
ith
-1, -
18 a
nd -4
6 m
olec
ular
ion
peak
s.
The
mol
ecul
ar io
ns o
f bot
h -1
and
-46
wer
e ch
arac
teri
zed
by th
e H
PLC
fra
ctio
nati
on fo
llow
ed b
y FA
B m
ass
spec
tra
and
amin
o ac
id c
ompo
sitio
ns.
The
-1 m
olec
ular
ion
was
con
clud
ed to
be
due
to c
leav
age
at a
mid
o bo
nds
inst
ead
of
pept
ide
bond
s w
hich
inte
rrup
t th
e fu
rthe
r deg
rada
tion
. T
he -
46 m
olec
ular
mas
s io
n ar
e du
e to
dec
arbo
xyla
tinn
and
al
so s
topp
ing
the
furt
her s
ucce
ssiv
e deg
rada
tion
. T
he u
nrec
over
able
- 18
ion
peak
s ar
e du
e tu
nit
rile
form
atio
n fr
om
Asn
and
Gin
resi
dues
, ri
ng-f
orm
atio
n of i
nter
nal
Asp
and
Glu
and
the
dehy
drat
ion
of S
er in
to d
ehyd
roal
anin
e.
The
se
are
pred
icte
d fr
om a
min
o ac
id c
ompo
sitio
ns a
nd a
ppea
ranc
es a
nd d
isap
pear
ance
s of
-18
ion
peak
s ac
cord
ing
to t
he
proc
ess o
f the
deg
rada
tion
. T
he -
18 p
eak
is d
ue to
oxa
zolo
ne fo
rmat
ion
at th
e re
spec
tive
pep
tide
C-t
erm
ini
whi
ch is
re
cove
rabl
e by
the
wat
er v
apor
trea
tmen
t. T
he p
redi
ctio
n of
oxa
zolo
ne fo
rmat
ion
is s
uppo
rted
by
the
opti
cal i
som
ers
wit
h th
e id
entic
al c
ompo
sitio
ns w
hich
wer
e se
para
ted
by H
PLC
. T
he p
redi
ctio
n w
as a
lso
supp
orte
d by
the
form
atio
n of
resp
ectiv
e th
e es
ters
by
the
addi
tion
of p
ropa
nol t
o th
e de
grad
ed p
rodu
cts i
dent
ifie
d by
FA
B m
ass
spec
tra.
T
hese
ob
serv
atio
n su
gges
t the
pos
sibl
e re
acti
on in
term
edia
te t
o be
the
oxaz
olon
es o
f the
C-t
erm
ini.
1. A
. Tsn
gita
et
al.,
Che
m.
Len.
199
2, 2
35.
2.A
. Tsu
gita
et
al.,B
iolo
gica
l M
ass
Spec
trom
etry
pp
.242
San
-ei
(199
2).
3. A
. Tsn
gita
et a
l., M
etho
ds i
n P
rote
in S
eque
nce
Ana
lysi
s pp
.55-
62, P
lem
num
Pre
ss, N
ew Y
ork.
(199
3)
T2
PRO
TE
IN D
OC
KIN
G IN
TH
E A
BSE
NC
E O
F D
ET
AIL
ED
MO
LE
CU
LA
R S
TR
UC
TU
RE
S
llya
A. V
akse
r C
ente
r for
Mol
ecul
ar D
esig
n, W
ashi
ngto
n U
nive
rsity
, St
. Lou
is, M
O 6
3130
, USA
One
of t
he b
asic
pro
blem
s in
pro
tein
- l
igan
d re
cogn
itio
n is
whe
ther
thi
s pr
oces
s is
gen
eral
ly d
eter
min
ed b
y lo
cal
stru
ctur
al e
lem
ents
, or
whe
ther
the
re a
re a
lso
larg
e-sc
ale
mot
ifs
in t
he m
olec
ule
stru
ctur
es
whi
ch
faci
litat
e th
e co
mpl
ex f
orm
atio
n. B
eyon
d its
con
cept
ual
impo
rtan
ce,
this
pro
blem
is
dire
ctly
rel
ated
to
a pr
acti
cal
poss
ibili
ty
to m
ake
pred
ictio
ns
of p
rote
in -
lig
and
com
plex
con
figu
ratio
ns
in t
he a
bsen
ce o
f de
tail
ed s
truc
tura
l da
ta o
n th
e m
olec
ules
. In
the
com
puta
tiona
l pr
oced
ures
for
pre
dict
ing
the
stru
ctur
e of
pr
otei
n co
mpl
exes
, tw
o m
olec
ules
ar
e br
ough
t in
con
tact
at
mul
tiple
rel
ativ
e po
sitio
ns,
the
exte
nt
of
com
plem
enta
rity
(g
eom
etri
c an
d/or
ene
rgy)
at
the
surf
ace
of c
onta
ct is
ass
esse
d at
eac
h po
sitio
n, a
nd t
he
best
fits
are
i'el
riev
ed. W
e m
odif
ied
our
prev
ious
ly d
evel
oped
doc
king
pro
cedu
re (
1, 2
) in
ord
er to
dep
rive
m
olec
ules
of
any
str
uctu
ral
feat
ures
sm
alle
r th
an 7
A.
The
mod
ifie
d pr
oced
ure
was
app
lied
on v
ario
us
know
n pr
otei
n co
mpl
exes
tak
en f
rom
the
Bro
okha
ven
Prot
ein
Dat
a B
ank.
T
hese
are
0~
- [~
sub
units
of
hum
an d
eoxy
hem
oglo
bin,
tx
- [~
sub
units
of
hors
e m
ethe
mog
lobi
n, a
spar
tic p
rote
inas
e -
pept
ide
inhi
bito
r, tr
ypsi
n -
tryp
sin
inhi
bito
r, ch
ymot
ryps
in -
ovom
ucoi
d th
ird
dom
ain,
and
Fab
fra
gmen
t -
lyso
zym
e. I
n al
l ca
ses,
exc
ept a
ntig
en -
antib
ody,
a p
rono
unce
d tr
end
tow
ards
the
corr
ect c
ompl
ex c
onfi
gura
tion
was
cle
arly
in
dica
ted
and
the
real
bin
ding
site
s w
ere
pred
icte
d. T
he d
istin
ctio
n be
twee
n th
e pr
edic
tion
of th
e an
tige
n -
anti
body
com
plex
and
the
oth
er m
~lec
ular
pai
rs m
ay r
efle
ct i
mpo
rtan
t di
ffer
ence
s in
the
pri
ncip
les
of
com
plex
form
atio
n. T
he r
esul
ts s
tron
gly
sugg
est t
he u
se o
f ou
r re
cogn
ition
pro
cedu
re fo
r pra
ctic
al d
ocki
ng
stud
ies
whe
n th
e de
tail
stru
ctur
e of
the
mol
ecul
es i
s la
ckin
g.
1.
E. K
atch
alsk
i-K
atzi
r et
al.,
Pro
c. N
atl.
Aca
d. S
ci. U
SA, 8
9, 2
195
(199
2).
2.
I.A
. V
akse
r, C
. Afl
alo,
in
pres
s (1
994)
.
V1
AC
ET
YL
-CoA
-CA
RB
OX
YL
ASE
: R
EG
IST
RA
TIO
N O
F A
CT
IVE
PO
LY
ME
R
CO
MP
LE
X
V.V
. V
elik
od
vo
rsk
aia,
A.G
. G
abib
ov
an
d A
.G.
Rab
ink
ov
, V
.A.
En
gel
har
dt
Inst
itu
te
of
Mo
lecu
lar
Bio
logy
, R
AS
, M
osc
ow
, R
uss
ia
Th
e p
roce
ss
of
the
reg
ula
tio
n
of
enzy
me
acti
vity
b
y
asso
ciat
ion
-dis
soci
atio
n,
of
its
sub
un
its,
eff
ecte
d b
y c
itra
te i
ons,
an
d b
y p
ost
-tra
nsl
atio
n m
od
ific
atio
n w
as i
nv
esti
gat
ed.
We
hav
e d
evel
op
ed
a n
ov
el
tech
niq
ue
bas
ed
on
th
e sp
ectr
ofl
ou
rem
etri
c an
d
ligh
t-
scat
teri
ng
stu
die
s o
f th
is e
qu
ilib
riu
m.
Th
is a
pp
roac
h a
llo
wed
us
to d
evel
op
qu
anti
tati
ve
esti
mat
ion
o
f th
e in
flu
ence
o
f p
ost
-tra
nsl
atio
n
mo
dif
icat
ion
s (p
ho
sph
ory
lati
on
) o
n
enzy
mat
ic a
ctiv
ity.
It
was
det
ecte
d t
hat
, af
ter
acti
vat
ion
of
Ace
tyl-
Co
A-c
arb
ox
yla
se b
y
sod
ium
ci
trat
e (a
llo
ster
ic
reg
ula
tor)
, th
e fl
uo
resc
ence
sp
ectr
um
o
f en
zym
e al
on
g w
ith
li
ght
scat
teri
ng
of
enzy
me
solu
tio
n u
nd
erg
oes
ch
ang
es.
We
sho
wed
th
at t
he
acti
vat
ion
p
roce
ss
con
nec
ted
w
ith
p
oly
mer
izat
ion
co
uld
b
e st
ud
ied
u
sin
g
men
tio
ned
p
hy
sico
- ch
emic
al
met
ho
ds.
T
he
kin
etic
co
nst
ants
o
f p
roce
ss
was
es
tim
ated
u
sin
g
dev
elo
ped
m
eth
od
s.
Ref
eren
ces:
1.
Eu
r. J
. B
ioch
em.
193,
35
1-3
53
(1
99
0)
2.
Bio
chem
istr
y 5
5,
1018
-102
3 (1
990)
. 3.
P
roc.
Ru
s. A
cad
. S
ci.
5, 2
2-2
5 (
1993
).
V2
Oq
ST
RU
CT
UR
E D
ET
ER
MIN
AT
ION
BY
NM
R O
F T
HE
NU
CL
EA
SE
IN
HIB
ITO
R
PR
OT
EIN
IM
8
Ten
nie
Vid
eler
I , M
ich
ael
Osb
orn
e 2,
Geo
ffre
y M
oo
re 2
, R
ich
ard
Jam
es I
and
Col
in
Kle
anth
ous
1 1 S
choo
l of
Bio
logi
cal
Sci
ence
s, U
nive
rsit
y of
Eas
t A
ngli
a, N
orw
ich
, 2
Sch
oo
l of
Ch
emic
al
Sci
ence
s, U
nive
rsit
y of
Eas
t A
ngli
a, N
orw
ich
Col
icin
E8
(Col
E8)
is
one
of
four
clo
sely
rel
ated
pla
smid
-en
cod
ed b
acte
rial
DN
ases
wh
ich
ar
e ex
pres
sed
as p
art
of t
he S
OS
res
pons
e sy
stem
in
som
e st
rain
s of
Esc
her
ich
ia c
olt.
Cel
ls
expr
essi
ng C
olE
8 p
rote
ct t
hem
selv
es f
rom
its
tox
ic a
ctiv
ity
by t
he c
oord
inat
e sy
nthe
sis
of a
sm
all,
hig
hly
spec
ific
inh
ibit
or p
rote
in,
call
ed t
he i
mm
un
ity
pro
tein
, Im
8 (
MW
9,5
83 D
).
IDE
NT
IFIC
AT
ION
OF
DIS
TIN
CT
PR
OT
EIN
AS
E K
CL
EA
VA
GE
SIT
ES
IN T
WO
ST
RA
INS
OF
SC
RA
FIE
PR
P B
Y C
HE
MIC
AL
ISO
LA
TIO
N O
F U
NIQ
UE
SE
QU
EN
CE
BY
O
RT
HO
PHT
AL
AL
DE
HY
DE
AN
D B
Y C
OM
PAR
ISO
N O
F N
-TE
RM
INA
L S
EQ
UE
NC
E M
APS
OF
PRP
FRA
GM
EN
TS
]ane
H. W
alen
t 1 , R
icha
rd B
esse
n 2, D
ick
Mar
sh 3,
Ron
ald
L. N
iece
1 1N
AP
Faci
lity,
Uni
v. W
isc.
Bio
tech
. Cen
ter,
Uni
v. W
isc.
, Mad
ison
, WI,
2NIH
, NIA
ID, R
ocky
M
ount
ain
Lab
s, H
amil
ton,
MT,
3D
ept.
An.
HIt
h. B
iom
ed. S
ci.,
Uni
v. W
isc.
, Mad
ison
, W
l
The
scr
apie
neu
ropa
thog
en is
thou
ght t
o ar
ise
thro
ugh
post
tran
slat
iona
l pro
cess
ing
of c
ellu
lar p
rion
pr
otei
n (P
rp C
) int
o in
fect
ious
pri
on a
myl
oid
prot
ein
(prp
S~.
prP
C a
nd P
rP Sc
hav
e id
entic
al
prim
ary
amin
o ac
id se
quen
ce,
and
stru
ctur
al st
udie
s ind
icat
e tha
t the
alt
erat
ion
of
seco
ndar
y/te
rtia
ry st
ruct
ure
is re
spon
sibl
e fo
r dif
fere
nces
in
biol
ogic
al a
ctiv
ity o
f PrP
isof
orm
s. T
he
pnes
ont s
tudi
es d
emon
stra
te di
ffer
ence
s in
pro
tein
con
form
atio
n of
two
stra
ins o
f Prl
'Sc,
term
ed D
Y
Pro
duct
ion
of a
spe
cifi
c in
hibi
tor
of a
ny
of
the
othe
r th
ree
clos
ely
rela
ted
coli
cins
wil
l no
t an
d FI
Y, w
hich
exh
ibit
sign
ific
ant
diff
eren
ces
in in
fect
ivity
. H
Y P
rP Sc
and
DY
PrF
Sc w
ere
trea
ted
prot
ect
the
cell
aga
inst
the
DN
ase
act
ivit
y o
f C
olE
8,
desp
ite
the
60
% h
om
olo
gy
bet
wee
n t
he
�9 W
ith p
rote
inas
e K
to g
ener
ate
dist
inct
am
yloi
d co
res d
iffe
rent
ially
sen
sitiv
e to
pro
teol
ysis
. im
mu
nit
y p
rote
ins.
We
are
inte
rest
ed i
n th
e st
ruct
ural
fea
ture
s de
term
inin
g th
e hi
gh
spec
ific
ity
and
the
very
tig
ht b
indi
ng;
esti
mat
ed d
isso
ciat
ion
con
stan
t is
<1
0 -1
3 M
.
The
sec
on
dar
y s
truc
ture
of
one
of t
he r
elat
ed i
mm
un
ity
pro
tein
s, l
m9
, ha
s be
en
deli
neat
ed a
nd t
erti
ary
stru
ctu
re m
odel
s ar
e cu
rren
tly
bein
g bu
ilt.
Mul
ti-
dim
ensi
on
al N
MR
ex
peri
men
ts h
ave
been
per
form
ed o
n 15
N l
abel
led
Im9
boun
d to
unl
abel
led
cofi
cin.
In
com
bina
tion
wit
h si
te d
irec
ted
mut
agen
esis
and
fus
ion
expe
rim
ents
thi
s ha
s in
dica
ted
at l
east
on
e si
te i
nvol
ved
in b
indi
ng o
n th
e im
mu
nit
y p
rote
in.
Co
mp
aris
on
of
an i
ndep
ende
ntly
de
term
ined
Ira
8 st
ruct
ure,
the
pro
gres
s of
whi
ch w
ill
be r
epor
ted,
wil
l re
veal
bot
h th
e st
ruct
ural
bas
is f
or t
he t
ight
bin
ding
bet
wee
n th
e to
xin
and
its
inhi
bito
r an
d th
e hi
gh
spec
ific
ity.
Tru
ncat
ed P
rP Sc
wer
e an
alyz
ed b
y SD
S-PA
GE
and
tran
sfer
red
to P
VD
F fo
r ana
lysi
s by
Edm
an
degr
adat
ion.
Alt
houg
h 4-
5 pr
otei
ns in
HY
and
DY
PrP
Sc w
ere
sequ
enci
ng, b
ased
on
the
excl
usiv
e pr
esen
ce a
nd a
bsen
ce o
f par
ticu
lar ~
=-I-
AA
sig
nals
in 8
of 1
2 cy
cles
ana
lyze
d, m
aps o
f N-t
erm
inal
se
quen
ce w
~e
dist
inct
for H
Y a
nd D
Y. T
he m
ajor
N-t
erm
inal
sequ
ence
in H
Y P
rp Sc
was
G-Q
-P-,
whe
reas
in D
Y P
rP Sc
it w
as G
/K~G
/P-G
/S -.
In tw
o ex
peri
men
ts,
inte
rnal
pep
tide
seq
uenc
e ne
ar th
e N
-ter
min
us o
f DY
PrP
Sc w
as is
olat
ed, i
n si
tu.
By
util
izin
g or
thop
htal
alde
hyde
to b
lock
non
-pro
line
N
-ter
min
i in
cyc
le 5,
or i
n cy
cles
11
and
14, w
e w
ere
able
to is
olat
e an
d an
alyz
e the
seq
uenc
es o
f m
ajor
pep
tide
s in
DY
PrP
Sc,
Thr
ee c
leav
age s
ites
wer
e id
entif
ied:
G
92 (m
ajor
), Q
98, K
101.
The
se
site
s wer
e no
t inc
lude
d in
repl
icat
e m
aps o
f N-t
erm
inal
PT
H-A
A si
gnal
s in
HY
Prp
Sc.
We
conc
lude
th
at th
ere
are
conf
orm
atio
nal d
iffe
renc
es b
etw
een
DY
and
HY
PrP
Sc w
hich
may
det
erm
ine
the
mol
ecul
ar b
asis
for s
trai
n di
ffer
ence
s in
neu
rode
gene
rati
ve ac
tivi
ty.
V3
WI
t~
LOCAL TIGHT PACKING OF HYDROPHOBIC GROUPS IN BETA-STRUCTURE.
Vtyurin N. Institute of molecular genetics,
Russian Academy of Science, Moscow, Russia,
The
purpose
of this study was to find some key feature of
beta-structure domains that may help to understand the structure
of majority beta-structure domains.
Space filling
modeling
of
beta-structure,
satistical
calculating
of
average
volume of
hydrophpobic groups of
known
globular
proteins,
analysis
of
hydrogen
bonds
grid
of
known
beta-domains were used in this
work.
It was shown that the tendency of hydrophobic
groups
to
form
a
local
tight packing on the surface of a beta-structure
sheet seems to be the principal reason for twisting of the beta-
sheet.
It was found that:
a) s
number of possible packing of
beta-sheets in beta-domains is very limited, b) the distribution
of amino acid side chains on the surface of beta-sheet is unique
and
allows
the
effective packing of hydrophobic groups on the
both side of beta-sheet.
The principle of local tight packing of hydrophobic
groups
in
beta-structure
can explain many important features of beta-
structure
domains
and
may
be
the
base
of
efficient
classification of known beta-structure domains.
References:
PROTEINS: Structure, Function, and Genetics
15, 62-70, 1993.
PROTEINS: Structure,
Function, and Genetics
(in press 1994).
V4
IDEN
TIFI
CAT
ION
OF
A C
LEAV
AGE-
SEN
SITI
VE R
EGIO
N IN
UPO
CO
RTI
N 1
BY
ISO
LATI
ON
OF
UN
IQU
E N
-TER
MIN
AL S
EQU
ENC
E BY
OR
THO
PHTA
LALD
EHYD
E Ja
ne H
. Wal
ent I
, Fr
anci
s H
. C. T
sao 2
, R
onal
d L.
Nie
ce 1
1NA
P F
acili
ty,
Uni
v. W
isc.
Bio
tech
. C
ente
r, U
niv.
Wis
c.,
Mad
ison
, W
l, 2D
ept.
Ped
iatr
ics
& P
erin
atal
Car
e, M
erite
r H
osp.
, M
adis
on,
Wl.
Two
Ca2
+-de
pend
ent
phoS
phol
ipid
bin
ding
pro
tein
s (P
LBP
) w
ere
purif
ied
from
rab
bit
lung
w
hich
st
imul
ate
Ca2
+-de
pend
ent
mem
bran
e fu
sion
in
surf
acta
nt v
esic
le a
ssem
bly
(1).
By
auto
mat
ed E
dman
deg
rada
tion,
the
36
kD l
ung
PLB
P w
as i
dent
ified
as
the
anne
xin,
lipo
cort
in 1
, an
d th
e 33
kD
fra
ctio
n w
as f
ound
to
cont
ain
seve
ral
prot
eins
(2)
. S
ince
pur
ified
36
kD P
LBP
was
ob
serv
ed t
o sp
onta
neou
sly
degr
ade
into
an
inac
tive
trun
cate
d fo
rm w
ith M
r 33
kD
, w
e ch
arac
teri
zed
this
fra
gmen
t in
ord
er t
o id
entif
y th
e cl
eava
ge s
ite.
The
3-4
prot
eins
obs
erve
d in
se
quen
cing
wer
e th
ough
t to
be
"rag
ged"
N-t
erm
ini,
or 3
3 kD
var
iant
s of
sto
rage
-sen
sitiv
e pe
ptid
e br
eaka
ge.
Bas
ed o
n re
lativ
e pm
ol s
igna
l per
cyc
le,
we
tent
ativ
ely
sort
ed 1
-2 m
ajor
se
quen
Ces
whi
ch a
lso
mat
ched
seq
uenc
e -
27-3
0 re
sidu
es f
rom
the
N-t
erm
inus
. Th
is w
ould
re
pres
ent
~ 3
kD d
elet
ion
from
the
N-te
rmin
us,
as a
ntic
ipat
ed.
To t
est
accu
racy
of
sequ
ence
pr
edic
ted
to b
e in
thi
s cl
eava
ge-s
ensi
tive
regi
on,
we
utili
zed
orth
opht
alal
dehy
de (
OP
A)
to b
lock
al
l N
-ter
min
i ex
cept
pro
line
in t
wo
cycl
es e
xpec
ted
to c
onta
in p
rolin
e (3
).
By
chem
ical
ly
isol
atin
g un
ique
seq
uenc
e fr
om t
he m
ixtu
re o
f ~
33 k
D f
ragm
ents
, w
e co
nfirm
ed t
he p
redi
cted
se
quen
ce a
nd i
dent
ified
the
reg
ion
susc
eptib
le t
o br
eaka
ge.
Furt
herm
ore,
we
wer
e ab
le t
o ve
rify
that
the
maj
or 3
3 kD
PLB
P p
urifi
ed f
rom
rab
bit
tung
(1)
was
not
a b
reak
dow
n pr
oduc
l of
36
kD
lipoc
ortin
1.
1.
Tsao
, F.
H.
C.
Bio
chim
. B
ioph
ys.
Act
s,
1045
, 29
(19
90).
2.
Ts
ao,
F. H
. C
., et
al.
Bio
chim
. B
ioph
ys A
cta
1081
, 14
1 (1
991)
. 3.
W
adsw
orth
, et
el.,
in
Tech
niqu
es i
n Pr
otei
n C
hem
istr
y, R
. H
. A
ngel
etti,
(E
d.)
Aca
dem
ic P
ress
, S
an
Die
go p
p. 6
1-68
(1
992)
.
W2
=.
f'b
Puri
fica
tion
and
Gen
eral
Cha
ract
eriz
atio
n of
Dim
ethy
lall
yl T
rypt
opha
n Sy
ntha
se
Hon
g W
ang;
C.
Dal
e Po
ulte
r;
Che
mis
try
Dep
artm
ent,
Uni
vers
ity
of
Uta
h.
The
fir
st p
athw
ay-s
peci
fic
step
in
the
bios
ynth
esis
of
ergo
t al
kalo
ids
in C
lavi
ceps
is
the
alky
latio
n o
f L
-try
ptop
han
at C
(4)
posi
tion
by
dim
ethy
lall
yl d
ipho
spha
te.
The
enz
yme
cata
lyze
d th
is r
eact
ion
is a
pre
nyltx
ansf
eras
e k
no
wn
as
Dim
ethy
lall
yl T
rypt
opha
n S
ynth
ase
( D
MA
T S
ynth
ase)
. D
MA
T s
ynth
ase
has
been
pur
ifie
d to
hom
ogen
eity
fro
m t
he s
ourc
e o
f C
lavi
ceps
pur
pure
a, A
TC
C s
trai
n 26
245.
The
met
hod
of
puri
fica
tion
inc
lude
s am
mon
ium
su
lfat
e pa
rtit
ion
and
a co
mbi
nati
on o
f io
n-ex
chan
ge c
hrom
atog
raph
y, c
hrom
atof
ocus
ing
and
I ge
l fi
ltrat
ion
chro
mat
ogra
phy.
The
nat
ive
enzy
me
appe
ars
as a
hom
odim
er w
ith
mo
no
mer
siz
e o
f 90
Kd.
The
gen
eral
cha
ract
eriz
atio
n o
f th
e en
zym
e w
as p
erfo
rmed
by
usin
g ra
dioc
hem
ical
as
say.
The
N-t
erm
inal
of
this
enz
yme
was
blo
cked
fr
om s
eque
ncin
g. D
iges
tion
of
the
enzy
me
wit
h pr
otea
se w
as u
sed
to g
et i
nter
nal
amin
o ac
id s
eque
nces
to
initi
ate
the
isol
atio
n o
f th
e ge
ne f
or t
his
enzy
me.
PRO
CES
SIN
G O
F TH
E M
ATU
RE
HIV
-1
NU
CLE
OC
APS
ID P
RO
TEIN
BY
THE
VIR
AL
PRO
TEA
SE
Ew
ald
M.
Won
drak
, A
lan
R.
Kim
mel
and
Joh
n M
. L
ouis
La
bora
tory
of
Cel
lula
r and
Dev
elop
men
tal
Bio
logy
, NID
DK
, NIH
, Bet
hesd
a, M
D 2
0892
In h
uman
im
mun
odef
icie
ncy
viru
s, R
NA
sel
ectio
n an
d pa
ckag
ing
duri
ng a
ssem
bly
invo
lves
the
tw
o cy
stei
ne a
rray
s (C
ys-X
2-C
ys-X
4-H
is-X
4-C
ys; C
CH
C)
in t
he n
ucle
ocap
sid
(NC
) pr
otei
n w
hich
for
m
retr
ovir
al-t
ype
zinc
fing
ers.
W
e de
mon
stra
te f
or t
he f
irst
tim
e th
e cl
eava
ge w
ithin
the
firs
t cy
stei
ne
arra
y (b
etw
een
Phe-
16 a
nd A
sh-1
7) o
f th
e N
C p
rote
in (
1) b
y th
e vi
ral
prot
ease
. T
he r
eact
ion
is
char
acte
rize
d by
a k
cat/K
m o
f 2.
2 m
M'~
min
"~ a
nd i
s in
hibi
ted
by s
peci
fic
aspa
rtic
aci
d pr
otea
se
inhi
bito
rs.
In th
eir
nativ
e st
ate,
the
CC
HC
arr
ays
are
held
in
a st
able
and
con
stra
ined
con
figu
ratio
n by
coo
rdin
ated
zin
c io
ns,
such
tha
t th
e N
C p
rote
in i
s no
t a
subs
trat
e fo
r th
e pr
otea
se.
How
ever
, re
mov
al o
f zi
nc i
ons
by c
hela
ting
agen
ts r
esul
ts i
n a
rela
xed
conf
orm
atio
n th
at i
s su
scep
tible
to
clea
vage
. In
con
tras
t, 3-
nitr
osob
enza
mid
e (2
) an
d cu
pric
ion
s th
at m
edia
te o
xida
tive
atta
ck
on t
he
cyst
eine
thio
late
res
idue
s of
the
arra
y ca
use
disu
lfid
e bo
nd f
orm
atio
n (3
) an
d re
leas
e zi
nc,
rend
er t
he
NC
pro
tein
res
ista
nt to
cle
avag
e. T
hese
res
ults
impl
y th
at p
rote
ase
inhi
bito
rs a
nd 3
-nitr
osob
enza
mid
e,
whi
ch i
mpa
ir e
arly
vir
al
infe
ctio
n, b
lock
an
esse
ntia
l re
gula
tory
ste
p th
at r
equi
res
NC
pro
tein
pr
oces
sing
by
the
vira
l pr
otea
se.
An
unde
rsta
ndin
g of
the
mec
hani
sm o
f zin
c re
mov
al i
n vi
vo a
nd/o
r in
hibi
tion
of th
e N
C p
rote
in p
roce
ssin
g m
ay h
elp
in d
esig
ning
nov
el in
hibi
tors
for
eff
ectiv
e an
ti-vi
ral
ther
apy.
1. K
. Sa
kagu
chi e
t al~
, Pre
e.N
atl.A
cad.
Sci.U
SA 9
0, 5
219
(199
3).
2. W
.G. R
ice e
t al~
, Pro
c.N
atI.A
cad.
Sci.U
SA 90
, 972
1 (1
993)
. 3.
J.R
. Cas
as-F
inet
et a
im, A
nnua
l Bio
phys
ical
Soc
iety
Mee
ting,
Was
hing
ton,
DC
, Feb
14-
18 (1
993)
.
NI
W3
A R
API
D S
CR
EE
NIN
G S
TR
AT
EG
Y F
OR
TH
E I
DE
NT
IFIC
AT
ION
OF
MO
DIF
IED
PE
PTID
E
FRA
GM
EN
TS
OF
GL
UT
AM
INE
SY
NT
HE
TA
SE U
SIN
G T
HE
HE
WL
ET
T-P
AC
KA
RD
M
AL
DI-
TO
F SY
STE
M
Scot
R. W
einb
erge
r, L
ynn
M. C
hake
l, A
lex
Apf
fel,
Julie
Sah
akia
n, C
had
G. M
iller
Pr
otei
n C
hem
istr
y Sy
stem
s, H
ewle
tt-Pa
ckar
d C
o., P
ale
Alto
, CA
943
04
Ghi
tam
ine s
ynth
etas
e is
ren
dere
d en
zym
atic
aUy i
nact
ive
via
a si
te-s
peci
fic o
xida
tion.
MA
LD
I-T
OF
mas
s sp
ectr
omet
ry co
mbi
ned w
ith p
rote
olyt
ie an
d ch
emic
al d
iges
tion
and
HPL
C f
ract
iona
tion
is d
emon
stra
ted
to f
acili
tate
the
rapi
d id
entif
icat
ion
and
isol
atio
n of
oxi
dize
d fr
agm
ents
. In
vit
ro C
NB
r, tr
ypsi
n an
d en
dopr
otei
nase
Lys
C d
iges
ts o
f nat
ive
and
oxid
ized
glu
tam
ine
synt
heta
se (b
oth
redu
cexl
/alk
ylat
ed an
d no
n-re
duce
d/al
kyla
ted)
wer
e sc
reen
ed b
y M
AL
DI-
TO
F M
S an
d co
mpa
red
to d
eter
min
e if
ions
with
m
olec
ular
wei
ght d
iffe
renc
es d
ue to
mod
ific
atio
n by
oxid
atio
n ar
e ob
serv
able
. M
olec
ular
wei
ght
diff
eren
ces f
or th
e pe
ptid
e fr
agm
ents
con
tain
ing H
is-2
69 w
ere
inde
ed o
bser
ved
betw
een
nativ
e an
d ox
idiz
ed g
luta
min
e sy
nthe
tase
dig
ests
. M
olec
ular
wei
ghts
wer
e de
term
ined
with
mas
s ac
cura
cies
of+
/-
0.02
% re
lativ
e to
the
pred
icte
d va
lues
. Su
bseq
uent
par
tial f
ract
iona
tion
by H
PLC
and
MA
LD
I-T
OF
anal
ysis
iden
tifie
d th
e ch
rom
atog
raph
ic el
utio
n tim
es o
f the
mod
ifie
d pr
otei
n fr
agm
ents
.
W5
W4