kinetics and thermodynamics of amyloid fibril formation ron wetzel university of tennessee

Kinetics and Thermodynamics of Amyloid Fibril Formation

Ron Wetzel

University of Tennessee

Energetics of Amyloid Fibril Formation

Fibril assembly equilibria and G fibril elongation

- Aβ(1-40) amyloid fibrils (Alzheimer’s disease) - polyglutamine amyloid (Huntington’s disease)

Kinetics of nucleated growth polymerization and G of nucleus formation

- polyglutamine amyloid

Thermodynamics of Amyloid Fibril Formation

• Some amyloidogenic mutations work by weakening native structure

- transthyretin

- Ig light chain

• local sequence also affects amyloidogenicity through fibril packing effects

Nfibril fibril

N

Time (days)

Aβ Amyloid Fibril Formation

lag phase

Cr

[Mo

no

mer

], μ

M

0

5

10

15

20

25

30

0 2 4 6 8 100

5

10

15

20

25

30

35

Th

T F

luo

resc

ence

(au

)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 300

20

40

60

80

100

Time (Hrs)

[Aβ

], μ

M

S26P mutant of Aβ(1-40)

The experimental Cr is the equilibrium position of fibril elongation

1. Unpolymerized Aβ at equilibrium: - chemically indistinguishable from initial - capable of making fibrils after concentration 2. Fibrils resuspended in buffer: - dissociate to the identical Cr position

Monomer + FibrilN FibrilN+1

Keq

Amyloid Fibril Elongation Thermodynamics

Keq = [FibrilN+1] / [FibrilN][Monomer]

Keq = 1 / [Monomer]

Keq = 1 / Cr

ΔG = - RT ln Keq

ΔG = - RT ln Keq = - RT ln (1 / 0.0000086)

ΔG = - 8.6 kcal/mol [wild type Aβ(1-40)]

CrMon

omer

rem

ain

ing,

μM

Time

-0.5

0

0.5

1

1.5

2

2.5

4 13 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 31 32 33 34 35 36 37 38 39 40

-0.5

0

0.5

1

1.5

2

2.5

4 13 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 31 32 33 34 35 36 37 38 39 40

*

Aβ(1-40) sequence position

ΔΔ

G,

kcal/

mol

Ala scan of Aβ(1-40) fibril elongation thermodynamics

ΔΔG(Ala – WT), kcal/mol

15-21

31-36

Ala scan of Aβ(1-40) fibril stability

Petkova et al., 2002

Guo et al., 2004

Positions 6 and 53 in parallel β-sheet in IgG binding protein G (β1)

6

53

Effect of Ala replacements in Aβ(1-40) amyloid and in Gβ1

ΔΔG(Ala – residue), kcal/mol

Mutation 18 19 20 31 32 36 6 / 53

Val Ala 1.25

Phe Ala 1.5

Ile Ala 1.65

Aβ(1-40 amyloid fibrils G (β1)

15-21

31-36

[Merkel et al., Structure 7, 1333 (1999) Williams et al., J. Mol. Biol. 357, 1283 (2006)]

inout



Mutation 18 19 20 31 32 36 6 / 53

Val Ala 1.3 1.0 1.25

Phe Ala 1.5

Ile Ala 1.65


15-21

31-36


inout



Mutation 18 19 20 31 32 36 6 / 53

Val Ala 1.3 1.0 1.25

Phe Ala 1.5 0.8 1.5

Ile Ala 1.65


15-21

31-36


inout



Mutation 18 19 20 31 32 36 6 / 53

Val Ala 1.3 1.0 1.25

Phe Ala 1.5 0.8 1.5

Ile Ala 2.0 1.0 1.65


15-21

31-36


inout

Pro scan of Aβ(1-40) fibril stability

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4 6 9 12 1415161718 19202122 23242526 27282930 3132333435 36373839


ΔΔ

G,

kcal/

mol

ΔΔG(Pro – WT), kcal/mol

[Williams et al., J. Mol. Biol. 335, 833-842 (2004)]

How Does Proline Destabilize β-Sheet?

• Backbone Effects - no N-H proton: lost H-bond - loss of planarity in extended chain

• Side Chain Packing Effects - Pro “side chain” is compact loop that does not extend far out of plane

Ala-edited Pro scan of Aβ(1-40) fibril stability

ΔΔG(Pro – Ala), kcal/mol


ΔΔ

G, k

cal/m

ol

-1

-0.5

0

0.5

1

1.5

2

2.5

4 14 15 16 17 18 19 20 21 22 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

[Williams et al., J. Mol. Biol. 357, 1283 (2006)]

ΔΔG values for Pro vs. Ala replacement in β-sheetGlobular Protein (Gβ1) vs. Amyloid (Aβ)

Gβ1 position ΔΔG, kcal/mol Source

53 > 4 Minor and Kim, Nature 367, 660 (1994)44 > 4 Minor and Kim, Nature 371, 264 (1994)


ΔΔ

G, k

cal/m

ol

-1

-0.5

0

0.5

1

1.5

2

2.5

4 14 15 16 17 18 19 20 21 22 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Amyloid

Globular Protein

T

Hydrogen-Deuterium Exchange Experiment

Deuterium- labeled fibrils

Processing Solvent (pH~2) - quench exchange - dissociate fibrils - efficient MS analysis

Afibrils

forward exchange - D2O, pD = 7.5

back exchange - H/D mix, pH ~ 210% D2O

( D ) ( H )

Data Analysis

MassSpectrometer

[Kheterpal, Zhou, Cook & Wetzel, PNAS (2000)]

Protected Amide Hydrogens in Proline Mutant Fibrils


0

2

4

6

8

10

12

14

16

18

4 6 9 12 14 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 33 34 35 36 37 38 39 WT

Position of Pro replacement

Deu

teri

um

co

nte

nt

few

er H

-bon

dsm

ore

H-b

onds

Leu34->Pro, ΔΔG = only 1.5 kcal/mol destabilized …. but it also has 4 more H-bonds than WT


Results:

- Aβ(1-40) fibril growth tends to a reversible equilibrium position with a Keq and ΔG

- ΔΔGs from Ala mutations agree with data from parallel β-sheet in globular protein

… propagated structural changes suggest a fundamental difference from globular proteins

- some ΔΔG effects attributable to energy changes within the monomer ensemble

fibril

N

Conformational space

G

globular protein amyloidogenic peptide

N

U U

A1

A3A2

A4

CAG (polyglutamine) expanded repeat diseases

Disease Largest Normal Smallest Abnormal

Huntington’s 39 36

Kennedy’s 33 38

SCA-1 39 41

SCA-2 31 35

SCA-3 (MJD) 41 40

SCA-6 18 21

SCA-7 17 38

DRPLA 35 51

SCA-17 44 46

Polyglutamine flanking sequences in expanded CAG repeat disease proteins

AVAAAAVQQSTSQQATQGTS--LTPQPIQNTNSLSILEEQQR-Qn-

PPPPQPQRQQHPPPPPRRTR--RGEPRRAAAAAGGAAAAAAR-Qn-

AVARPGRAATSGPRRYPGPT--PRPHVSYSPVIRKAGGSGPP-Qn-

RDLSGQSSHPCERPATSSGA--SGTNLTSEELRKRREAYFEK-Qn-

PPPAAANVRKPGGSGLLASP--GCPRPACEPVYGPLTMSLKP-Qn-

HLSRAPGLITPGSPPPAQQN--YSTLLANMGSLSQTPGHKAE-Qn-

ETSPRQQQQQQGEDGSPQAH--GPRHPEAASAAPPGASLLLL-Qn-

HHGNSGPPPPGAFPHPLEGG--PSTGAQSTAHPPVSTHHHHH-Qn-

PPPPPPPPPPPQLPQPPPQA- MATLEKLMKAFESLKSF-Qn-

TBP (SCA17)

Ataxin 7 (SCA7)

CACNA1A (SCA6)

Ataxin 3 (SCA3)

Ataxin 2 (SCA2)

Ataxin 1 (SCA1)

Androgen Receptor (SBMA)

Atropin 1 (DRPLA)

Huntingtin (HD)

0 75 150 225 300

0

20

40

60

80

100

120

Lig

ht S

catt

erin

g

Hours

20 M Q28 monomer

20 M Q28 monomer + 1% Q28 aggregate

Lag phase aborted by seeding

Nucleation / Elongation

M N*k1

k-1

G

Reaction coordinate

Mk3

MMk2

M

N*

k4

N+1

N+2

N+1 N+2

Kn*

nucleation equilibrium constant

second order fibril elongation rate constant

= ½ Kn*k+2Cn*+2t2

[Qn] time

Nucleation Kinetics Analysis for Q47 Aggregation

time2 plots

slope = ½ Kn*k+2Cn*+2

-15

-14

-13

-12

-11

-4.9 -4.8 -4.7 -4.6 -4.5 -4.4 -4.3 -4.2 -4.1 -4 -3.9

log ([monomer], M)

log

(t2

slo

pe)

slope = n* + 2 = 2.87

n* = 0.87 ~ 1

log (½ Kn*k+2) = -0.7668

time2 (sec2)

[po

lyG

ln],

M (

x 10

6 )

7

12

17

22

27

32

37

0.0E+00 1.0E+08 2.0E+08 3.0E+08 4.0E+08 5.0E+08

80

85

90

95

100

105

110

[po

lyG

ln],

M (

x 10

6 )

+

nucleation

elongation

Mechanism of polyglutamine aggregation

Kn*

n* = 1 for Q28, Q36, Q47; Kn* increases from Q28 to Q36 to Q47

[Chen, Ferrone & Wetzel, PNAS (2002)]

Calculated Aggregation Kinetics Curves at Low Concentration

Q47 Q36 Q28

10-4

10-3

10-2

10-1

100

101

102

103

104

0

10

20

30

40

Years

Agg

rega

ted

Pep

tid

e (%

)[Qn] = 0.1 nM

31 141 1,273

= ½ k+2 Kn* c(n*+2) t2


Time (sec)

ln [

mo

no

mer

, M

]

-10.28

-10.26

-10.24

-10.22

-10.20

-10.18

-10.16

-10.14

0.0E+00 5.0E+03 1.0E+04 1.5E+04 2.0E+04 2.5E+04

Pseudo-first order kinetics of seeded polyGln elongation

[A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]

Fibriln + Monomer Fibriln+1

Rate = k+ [Fibril][Monomer] = kpseudofirst [Monomer]

k+ = kpseudofirst / [Fibril]

= ½ k+2 Kn* c(n*+2) t2

-0.7668 = log (½ Kn*k+2)

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 100 1 2 3 4 5 6 7 8 9 10

[biotinyl-polyglutamine], μM

fmo

l b

ioti

ny

l-p

oly

Gln

bo

un

d

+

Determination of Kn*

k+ = kpseudo1st / [aggregate] = 1.14 x 104 liters/mol-sec

-0.7668 = log (½ Kn*k+2)

Kn* = 2.6 x 10-9

ΔG = + 12.2 kcal/mol[A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]

nucleation

+

elongation

Mechanism of polyglutamine aggregation

Kn*

For Q47, Kn* = 2.6 x 10-9 (ΔGnucleation = + 12.2 kcal/mol)

k+

[A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]

Acknowledgments

Aβ Team PolyGln Team

Angela Williams Anusri BhattacharyyaShankari Shivaprasad Ashwani Thakur

Brian O’Nuallain Songming ChenIndu KheterpalEric Portelius

Frank Ferrone (Drexel Univ.) Trevor Creamer (Univ. Kentucky) Veronique Hermann (Univ. Kentucky)

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350

Time (hrs)

% M

on

om

erPolyproline dampens polyglutamine aggregation

Q40

Q40P10

P10Q40

H2NKKQ

40CKKCOOH

|SCH

2CONHG

3P

10 COOHKK

[A Bhattacharyya et al., J. Mol. Biol. 2006]

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350

Time (hrs)

% M

on

om


Q40

Q40P10

Cr = 4.5 μM

Cr ≤ 50 nM

ΔΔG ≥

3 kcal/mol

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350

Time (hrs)

% M

on

om


Q40

Q40P10

P10Q40

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350

Time (hrs)

% M

on

om


Q40

Q40P10

P10Q40

H2NKKQ

40CKKCOOH

|SCH

2CONHG

3P

10 COOHKK

0

2

4

6

8

10

12

0 100 200 300 400

Time (hrs)

[Mo

no

me

r],

µM

Is the Plateau a Real Thermodynamic Cr?

Q40

Q40

Q40P10

Q40P10


A Conformational Correlate to the P10 Connectivity Effect on Aggregation

35°C - 5°C difference spectra


A Possible Basis of the OligoProline Effect

Conformational Space

G

fibril

aggregation- incompetent monomer

aggregation- competent monomer

ΔG

Transportability of the P10 Effect

Peptide C r, μM

Aβ(1-40) 0.9 μM

Aβ(1-40)-P10 21.5 μM


Side Chain Packing by Disulfide Formation

HS

[O]HS

HS

SH

HS

SH

HS

HS

S

SH

HS

S

[S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)]

Stability of amyloid fibrils from various double Cys mutants of Aβ(1-40)

R-SH R-S-S-R

Cysteine mutants

0

0.5

1

1.5

2

2.5

3

17C-34C 17C-35C 17C-36C

ΔΔ

G (

kca

l/m

ol)

[S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)]

16

1718

1920

21

31

32

33

343536

15

Re

lati

ve

In

ten

sit

y

20-34+2

A

B

(c)

746 750 754

Mass/Charge

(d)35-40

+1

561 565 569

727

(a)1-40+6

A

B

723 731

(b)

1-19+5

461 465 469

Re

lati

ve

In

ten

sit

y

Mass/Charge

HX-MS with in-line pepsin: distribution of protected amide protons

[M. Chen, I. Kheterpal, K. D. Cook and R. Wetzel, unpublished]

M N*k1

k-1

Mel

k3

MelMel

k2 k4

G

Reaction coordinate

M

N*

N+1

N+2

N+1 N+2


N*

45

55

65

75

85

95

105

0 1 2 3 4 5 6

Time (hrs)

Rel

ati

ve

[Q47

]

Q47 Nucleation Kinetics in the Presence of Various Concentrations of Q20

2 M Q47 + [Q20 ], M

0

14

24

36

44

54

30

40

50

60

70

80

90

100

110

0 2 4 6 8

Time (hrs)

Re

lati

ve

[Q

47

]

Q47 Nucleation Kinetics in the Presence of other PolyGln Peptides

2 M Q47 + 20 M ….

No addnQ10

Q15

Q20

Q25

Q29

Q33

Q40

+

0

5

10

15

20

0 5 10 15 20 25 30

Time, mins

fmo

l b

ioti

n-Q

30

-10.3

-10.28

-10.26

-10.24

-10.22

-10.2

-10.18

-10.16

-10.14

0 5000 10000 15000 20000 25000

Time (sec)

ln [

mo

no

mer

, M]

Determination of Q47 fibril second order elongation rate constant

k+ = kpseudo1st / [growing ends]

k+ = 11,900 moles/liter-sec

How is amyloid formation initiated? Polyglutamine studies

There are no kinetically relevant intermediates in nucleation of simple polyGln peptides

Results:

- the nucleus for polyGln aggregation is an energetically unfavorable monomer

- repeat length dependent nucleation efficiency may help account for ages-of-onset

- Kn* for a Q47 peptide is ~ 10-9

- short polyGln peptides in the environment can enhance nucleation efficiency


M N*k1

k-1

G

Reaction coordinate

Mk3

MMk2

M

N*

k4

N+1

N+2

N+1 N+2

Kn*

nucleation equilibrium constant

second order fibril elongation rate constant

= ½ Kn*k+2Cn*+2t2

[Qn] time

16

1718

1920

21

31

32

33

34

3536

15

Side Chain Orientation and Packing Within the Aβ(1-40) Amyloid Fibril

[S. Shivaprasad, J.-T. Guo, Y. Xu and R. Wetzel, unpublished]

Side Chain Orientation by Cys Accessibility

SH

SH SH

I-CH2C(O)NH2

S-CH2C(O)NH2

Ala-edited Pro scan of Aβ(1-40) fibril stability

ΔΔG(Pro – Ala), kcal/mol


ΔΔ

G, k

cal/m

ol

-1

-0.5

0

0.5

1

1.5

2

2.5

4 14 15 16 17 18 19 20 21 22 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4 6 9 12 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 P2 P4

Proline Mutant

dd

G,

kcal

/mo

l

Amyloid Fibril Thermodynamics

WT DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVVP2 P PP4 P P P P


-0.5

0

0.5

1

1.5

2

2.5

3

3.5

19 20 38 39

Alanine mutation ΔΔGs adjust for hydrophobicity effects in Pro series

Proline - WT ΔΔG

Alanine - WT ΔΔG

Pro-Ala ΔΔG

Aβ Sequence Position

ΔG

mu

t –

ΔG

wt,

kcal/

mol

[AD Williams & R Wetzel, Ms. in preparation]

Additivity in Alanine mutation ΔΔGs

16

1718

1920

21

31

32

33

343536

15

0

0.5

1

1.5

2

2.5

17 34 17+34 17/34 17 25 17+27 17/27

Ala Mutants

ΔΔ

G,

kc

al/

ml

[AD Williams & R Wetzel, Ms. in preparation]

Aβ(1-40) monomer seeded with Aβ(1-40) or IAPP fibrils

0

2

4

6

8

10

0 1 2 3 4Time (hrs)

0

0.1

0.2

0.3

0.4

0.5

0.6

0 1 2 3 4Time (hrs)

All experiments with 10 nM biotinyl-Aβ

Fm

ol b

ioti

nyl-

Aβ

Fm

ol b

ioti

nyl-

Aβ

Aβ amyloid fibrils on plate

IAPP amyloid fibrils on plate

IAPP fibrils are only 1-2% efficient, compared with Aβ, in seeding Aβ elongation.

Collagen on plate

[O’Nuallain, Williams, Westermark & Wetzel, J. Biol. Chem. 279, 17490-17499 (2004)]

Rates of A Elongation with Various Amyloid Fibrils as Seeds

Seed Fibril Elongation Rate (fmol/hour) Relative Efficiency

A 7.5 ± 1.1 100 %

IAPP 0.086 ± 0.01 1.1

Ig light chain LEN (1-30) 0.019 ± 0.001 0.3

Ig light chain VL JTO5 0.042 ± 0.006 0.6

2-microglobulin 0.014 ± 0.001 0.2

Ure2p 0.069 ± 0.001 0.9

Polyglutamine Q30 0.44 ± 0.01 5.9

Collagen 0.0075 ± 0.001 0.1

Ovalbumin, reduced/alkylated 0.009 ± 0.003 0.1

[O’Nuallain, Williams, Westermark & Wetzel, J. Biol. Chem. 279, 17490-17499 (2004)]

Wavelength(nm)

Random coil to -sheet transition in a Q42 peptide incubated at pH 7, 37 °C

-10000

-5000

0

5000

10000

15000

20000

200 220 240 260

[]

deg

ree

cm2 d

mol

e-1

T = 0 hrs

T = 217 hrs

T = 86 hrs

T = 45 hrs


Fractionation of an Incomplete Aggregation Reaction

200 220 240 260

-10000

-5000

0

5000

10000

15000

20000

Wavelength(nm)

[]

deg

ree

cm2 d

mol

e-1

aggregation time point (86 hrs)

resuspended pellet

supernatant

supernatant plus pellet spectra

No evidence for stable, -sheet structure in the non-aggregated fraction

14

1210

8

6

4

2

40

38

16

18

282624

22

20

36

34

32

30

14

1210

8

6

440

38

40

38

16

18

282624

22

20

36

34

32

30

A Working Model for the Aβ(1-40) Fibril


[Guo, J.T., Wetzel, R. and Xu, Y., Proteins (2004) In press.]

0 50 100 150 200 250

0

20

40

60

80

100

Hours

% A

ggre

gate

For

mat

ion

Aggregation of a Q42 Peptide Monitored by Four Parameters

-sheet formation proceeds in parallel with aggregation

HPLC insolubles

Thioflavin T fluoresence

-sheet (CD)

Light scattering

Protein Deposition in Human Disease

• Amyloid Plaques (Alzheimer’s)• Amyloid Angiopathy (microvasculature)• Neurofibrillary Tangles (Alzheimer’s; tauopathies)• Lewy Bodies (Parkinson’s; Lewy Body Dementia)• Polyglutamine aggregates (Huntington’s)• Rosenthal Fibers (astrocytes)• Prion Diseases• SOD aggregates (ALS)

• Amyloid (heart, kidney, liver, lungs, peripheral nerves, spleen, skin) - serum amyloid A - transthyretin - Ig light chain - islet amyloid polypeptide (IAPP) - β2-microglobulin• Z-form 1-Antitrypsin Deposition (liver)• Inclusion Body Myositis (muscle)• Mallory Bodies (liver)

BRAIN

PERIPHERY

0

5

10

15

20

25

30

0 0.5 1 1.5 2 2.5 3 3.5 4Time (h)

Th

T f

luo

resc

ence

or

[Aβ

] (μ

M)

Seeded amyloid growth from Aβ(1-40)

ThT

[Aβ(1-40)]

Cr

0

5

10

15

20

25

30

0 0.5 1 1.5 2 2.5 3 3.5 4Time (h)

Th

T f

luo

resc

ence

or

[Aβ

] (μ

M)


ThT

[Aβ(1-40)]

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6

Time (hrs)

Th

T f

luo

res

ce

nc

e

Seeded amyloid growth from Aβ(1-40) concentrated from Cr plateau

0

5

10

15

20

25

30

0 0.5 1 1.5 2 2.5 3 3.5 4Time (h)

Th

T f

luo

resc

ence

or

[Aβ

] (μ

M)


ThT

[Aβ(1-40)]

Aβ(1-40) fibril dissociation to equilibrium

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50 60

Time (hrs)

[Aβ

] (μ

M)

0.5-day fibrils

20-day fibrils

CAG REPEAT LENGTHS IN HUNTINGTON’S DISEASE

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 4525 26 27 2824

penetrance

Repeat Length Dependence of Age of Onset in Huntington’s Disease

[Courtesy Marcy MacDonald]

log C

-0.3 0.0 0.3 0.6 0.9 1.2 1.5 1.8

-5

-4

-3

-2

-1

0

Q28

Q36

Q47

Concentration Dependence of Nucleation Kinetics

log

[½ k

+2 K

n* c

(n*+

2)]

slope = n* + 2


0

5

10

15

20

25

30

35

40

0 100 200 300 400 500 600

Time (hrs)

Mo

no

me

r (u

M)

Q45

PGQ9

PGQ8

PGQ7

0

5

10

15

20

25

30

0 50 100 150 200

Time (hrs)

Mo

no

mer

(u

M)

PGQ9

PDGQ9

PolyGln Aggregate Structure

0

5

10

15

20

25

30

35

0 100 200 300 400

Time (hrs)

Mo

no

me

r (u

M) PGQ9(P

2)

Q15PQ26

PGQ9

Q45

PGQ9 PG PG PG

PG PG PGPGQ9(P2) P

PQ15PQ26

PolyGln Aggregate Structure

PG

PG

PG

PG

PG

PG

N

N

C

C

PG

PG

PG

PGG

PG

P

N

N

C

C

Anti-parallel -sheet model Parallel -helix model

Aggregation-competentmonomer

Aggregation-incompetentmonomer

(polyproline type II helix??)

AggregateWetzel and Creamer labs

Wetzel lab

Computer simulations: Rohit Pappu, Washington University

Effect of flanking sequences on polyglutamine aggregate stability

Summary

• as predicted by theory, in vitro amyloid fibrils can achieve an equilibrium with monomer

• the position of this equilibrium is proportional to the free energy of fibril formation

• measurement of shifted equilibria allows quantitation of mutational effects

• amyloid fibrils exhibit a remarkable structural plasticity

• in ideal cases, aggregation kinetics can be interpreted mechanistically

• the kinetic nucleus for polyglutamine aggregation is an alternatively folded monomer

• accumulated sequence changes strongly diminish cross-seeding efficiency

Mutagenesis and Kinetics/Thermodynamics in Globular Protein Structure

• studies on “natural” mutants of globular proteins (1970s) - Gary Ackers (human hemoglobin variants) - Mike Laskowski (ovomucoid variants)

• protein engineering approaches to globular protein folding stability (1984->) - Ron Wetzel (T4 lysozyme disulfide bonds) - Brian Matthews (T4 lysozyme point mutations) - Robert Matthews, Alan Fersht (folding kinetics)

• protein folding stability and amyloidogenicity (1993->) - Jeff Kelly (transthyretin / TTR amyloidosis) - Ron Wetzel (light chain FV domain / Ig light chain amyloidosis) - Chris Dobson (lysozyme amyloidosis)

• amyloid fibril assembly kinetics and thermodynamics….landscape continuity? - kinetics complicated by protofibrils and by secondary nucleation - can fibril formation reach true equilibrium positions in vitro?

Aggregation and Packing Interactions

[R. Wetzel, Trends Biotech. 12, 193-198 (1994)]

ACKNOWLEDGMENTS

UTMCK• Indu Kheterpal• Angela Williams• Shankaramma Shivaprasad• Israel Huff• Tina Richey• Kimberley Salone• Matt Sega• Brian Bledsoe• Valerie Berthelier• Lezlee Dice• Brian O’Nuallain• Anusri Bhattacharyya Mitra• Songming Chen• Wen Yang• Brad Hamilton• Ashwani Thakur• Geetha Thiagarajan• Roopa Kenoth• Merav Geva• Alex Osmand• Erica Johnson Rowe• Erin Newby

UGA• Juntao Guo• Ying Xu

UT Main Campus

• Maolian Chen• Erik Portelius• David Kaleta• Shaolian Zhou• Kelsey Cook

• Neil Whittemore• Rajesh Mishra• Engin Serpersu

• Guangyao Gao• Ying Chen• Peter Zhang

• Anna Gardberg• Chris Dealwis

• Liz Howell

• John Dunlap

Harvard Med• Hilal Lashuel• Peter Lansbury• Prasanna Venkatraman• Fred Goldberg

FUNDING: NIH (NIA, NINDS); Hereditary Disease Foundation

Cal Tech• Jan Ko• Susan Ou• Paul Patterson

Uppsala

• Per Westermark

Drexel • Frank Ferrone


• In globular proteins, some amyloidogenic mutations work by weakening native structure - transthyretin (Kelly) - Ig light chain (Wetzel)

• local sequence also affects amyloidogenicity through fibril packing effects

• simplest systems are where the starting monomer is in coil, ….. - no overlay of a stable native state - reasonable assumption that mutation minimally affects native state G

• ….. and where there is an easily and accurately measured Cr

• Results: - Aβ(1-40) fibril growth tends to an easily measured, reversible equilibrium position - ΔG = - 8.6 kcal/mol - ΔΔGs from Ala mutations agree with data from parallel β-sheet in globular protein - Ala-edited Pro scan reveals sequence segments in rigid structure, ….. - … but propagated structural changes in H-bonding complicate interpretation

Many Pro-destabilized Aβ(1-40) fibrils gain H-bonds


0

2

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6

8

10

12

14

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18

4 6 9 12 14 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 33 34 35 36 37 38 39 WT

Position of Pro replacement

Deu

teri

um

co

nte

nt

few

er H

-bon

dsm

ore

H-b

onds

Normal globular proteins generally have only one stable state

kinetics and thermodynamics of amyloid fibril formation ron wetzel university of tennessee

Documents

amyloid fibrils g

g fibril elongation

ala scan of a1

phe ala

ile ala

val ala

g1 g ala residue

effect of ala replacements