Solution to Gas PhaseSolution to Gas Phase

•• DNADNA–– Secondary StructureSecondary Structure–– QuadruplexQuadruplex FormationFormation

•• Protein ComplexesProtein Complexes–– pH dependencepH dependence–– NoncovalentNoncovalent interactionsinteractions

H+

H+

H+

H+

H+ M

H+

H+

H+

H+

H+ MH+

M(H+)n

Droplet after reduction size due to evaporation

“CoulombicExplosion”

Multiply Protonated

Analyte

Ionization Process

H+

H+

H+

H+

H+H+

H+H+

H+

H+M

M

1-10µm

H+

H+

H+

H+

Experimental Methods

Detector

m/z

MSIonFunnel

MASS SPECTRUM

N-ESI Source

Experimental Methods

Detector

m/z

MSIonFunnel

1 – 5 Torr HeE

MSDriftCell

IonFunnel

time

ARRIVAL-TIME DISTRIBUTION

MASS SPECTRUM

N-ESI Source

DNA

• Secondary Structure–AT Helix Stability

• Quadruplex Formation–Telomer

DNA Conformations

• A double stranded right-handed helix is a regular conformation adopted by both DNA and RNA in cells.

• An increasing number of results also point to the biological importance of alternate structures such as bulges, hairpins, branched junctions and quadruplexes

• TTAGGG repeats at ends of chromosomes play important role in cell life and cancer

• Quadruplexes are possible telomerase inhibitors for cancer treatment

• F. Rosu, V. Gabelica, C. Houssier, P. Colson, E. De Pauw Rapid Commun. Mass Spectrom. 2002, 16, 1729.

Observed triplexes and quadruplexes cationized by NH4+ in ESI-MS

MS/MS: triplex → duplex + antigene (similar to solution dissociation)

• T. Aggerholm, S.C. Nanita, K.J. Koch, R.G. Cooks J. Mass Spectrom. 2003, 38, 87

Observed “magic number” G-quartet adducts in ESI-MS

Quadruplexes in the Gas Phase

• S.A. Ho stadler, R.H. Griffey, Chem. Rev. 2001, 101, 377

Review of evidence that noncovalent RNA and DNA complexes are transferred from solution into the gas phase

G-Quartet (Quadruplex Unit)

N

N

N

NH

OH

N

H

H

NN

N N

H

O

HNHH

N

N

N

NH

OH

N

H

H

NN

NN

H

O

H N HH

G

G

G

G

G

G

G

G

G

G

G

G

5´ 3´

3´5´

Telomere Sequence dTTAGGGTTAGGG

2-strand system

12-mer

ESI Mass Spectrum of (dTTAGGGTTAGGG)

m/z

1100 1200 1300 1400 1500 1600

[single strand]3-

+ NH4[dimer]5-

+ NH4

time (µs)550 600 650 700 750

time (µs)550 600 650 700 750

σexpt = 773 Å2

Theoretical Structures of [dimer]5-

Quadruplex

σexpt = 773 Å2

Globular

σtheory = 780 Å2 σtheory = 748 Å2

dTTAGGGTTAGGGTTAGGGTTAGGG

intramolecular quadruplex

G

G

G

G

G

G

G

G

G

G

G

G

5´

3´

24-mer

m/z

1000 1100 1200 1300 1400 1500 1600

7-

6-

5-

ESI Mass Spectrum of (dTTAGGGTTAGGGTTAGGGTTAGGG)

arrival time (µs)550 600 650 700 750

arrival time (µs)550 600 650 700 750

arrival time (µs)550 600 650 700 750

σexpt = 773 Å2

Theoretical Structures of [single strand]5-

Quadruplex Globular

σexpt = 773 Å2

σtheory = 776 Å2σtheory = 746 Å2

Duplexes in the Gas Phase

• V. Gabelica and E. DePauw, Int. J. Mass Spectrom. 2002, 219, 151.

• P.D. Schnier, J.S. Klassen, E.F. Strittmatter and E.R. Williams,J. Am.Chem. Soc. 1998, 120, 9605-9613.

Higher Ea for complimentary duplexes and Ea correlated to –∆Hd in solutionEvidence of Watson-Crick pairing in vacuo

CID yields correlate with number of GC pairs and ∆Hdiss in solutionSuggests structure conserved in gas phase

• M. Rueda, S.G. Kalko, F.J. Luque, M. Orozco J. Am. Chem. Soc. 2003, 125, 8007

Gas-phase MD simulations indicate 12- and 16-mer duplexes retain major

conformational features as the double helix in aqueous solution

W. Fuller, et al

J. Mol. Biol. 1965, 12, 60

75% relative humidity

3 Common Helical Duplexes

A.H.J. Wang, et al

Nature 1979, 282, 680

CGCGCG in high salt conc.

A-Form B-Form Z-Form

R. Langridge, et al

J. Mol. Biol. 1960, 2, 19

92% relative humidity

d(AT)5 ATDs

450

Arrival Time (µs)

550 650 750 850 950

819 Å2

758 Å2

916 Å2

time (ps)

Cro

ss-s

ectio

n (Å

2 )

dAT 10-mer (A-form) 300K dynamics

σEXPT = 758, 819, 916 Å2

820 Å2

760 Å2

920 Å2

d(AT)5 ATDs

450

Arrival Time (µs)

550 650 750 850 950

σexp = 819 Å2

σ theory = 820 Å2σexp = 758 Å2

σ theory = 760 Å2

σexp = 916 Å2

σ theory = 920 Å2

ESI spray

dehydrate dehydrate

B-DNA(90% humidity)

A-DNA(75% humidity)

Solution vs. Solvent-Free Structures

Ion Funnel

920 Å2

760 Å2

820 Å2

DNA Summary

• DNA helices are observed in the gas phase• B-DNA → A-DNA transitions upon

dehydration• Quad structures conserved

– In absence of solvent– In absence of salt

Protein Complexes

Aβα-synuclein

Solution → Gas Phase

DAEFRHDSGYEVHHQKLVFF20AEDVGSNKGAIIGLMVGGVV40IA42

N CAβ

ER

Mem

bran

e

CytoplasmLumen

Amyloid β-proteinprecursor (AβPP)

β-Secretase

wild type sequence

γ -Secretase

Aβ42- F19PSubstitution by proline

Image courtesy of the Ronald Reagan Presidential Foundation, all rights reserved."

6 Acidic 3 Basic

Total charge: -3

N CAβ

ER

Mem

bran

e

CytoplasmLumen

Amyloid β-proteinprecursor (AβPP)

β-Secretase γ -Secretase

DAEFRHDSGYEVHHQKLVFF20AEDVGSNKGAIIGLMVGGVV40IA42

2

0800

M (-3)

D (-4)D (-5)

M (-4)

M (-5)T (-7)

1000 1200 1400 1600 1800 2000 2200 24000

100

%

m/z

2

0800

M (-3)

D (-4)D (-5)

M (-4)

M (-5)T (-7)

1000 1200 1400 1600 1800 2000 2200 24000

100

%

m/z

800 1000 1200 1400 1600 1800 2000 2200 24000

100

%

M (-3)

M (-4)

M (-5)

0800 1000 1200 1400 1600 1800 2000 2200 2400

0

100

%

M (-3)

M (-4)

M (-5)

0

Aβ42-wt

Aβ42-F19P

time (µs)550 650 750 850 950

time (µs)550 650 750 850 950

time (µs)1000600 700 800 900

time (µs)1000600 700 800 900

time (µs)550 650 750 850 950

time (µs)550 650 750 850 950

D

M1 M2

Radius of gyration (Å)

Cro

ss s

ectio

n (Å

2 )

Gas phase

550

600

650

700

750

800

850

900

950

1000

7 9 11 13 15 17 197

experimentexperiment

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA42

• Generate 100s of model structures using:Molecular mechanics (CHARMM22)Replica exchange algorithmImplicit solvent (GB/SA) and Gas Phase

• Calculate for each structure:Cross sectionRadius of gyration

• in collaboration with Andriy Baumketner and Joan Shea (UCSB)

Model structures of Aβ42-wt monomers

Radius of gyration (Å)

Cro

ss s

ectio

n (Å

2 )

Gas phase

550

600

650

700

750

800

850

900

950

1000

7 9 11 13 15 17 197

implicitwater

experimentexperiment

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA42

7 9 11 13 15 17 19

Radius of gyration (Å)

Cro

ss s

ectio

n (Å

2 )

dehydrated

550

600

650

700

750

800

850

900

950

1000

7

implicitwater

experimentexperiment

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA42

9 11 13 15 17 19

Radius of gyration (Å)

Cro

ss s

ectio

n (Å

2 )

Gas phase

implicitwater

experiment

dehydratedexperiment

550

600

650

700

750

800

850

900

950

1000

7

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA42

Dehydrate

Implicit water

F19 F19

Aβ- wt Structures

Dehydrate

Implicit water

F19 F19

Gas Phase

Aβ- wt Structures

F19

1 10 20 30 40MDVFMKGLS KAKEGVVAAA EKTKQGVAEA AGKTKEGVLY VGSKTKEGVV 50 60 70 80 90HGVATVAEKT KEQVTNVGGA VVTGVTAVAQ KTVEGAGSIA AATGFVKKDQ 100 110 120 130 140LGKNEEGAPQ EGILEDMPVD PDNEAYEMPS EEGYQDYEPE A

Amino-acid sequence of human α-synuclein. The seven imperfect repeats are underlined.

Parkinson’s Disease: α-synuclein

700 900 1100 1300 1500 1700 1900 2100 2300 2500

pH 7

pH 2.5

α-synuclein

-9(M)

-12(M)

-13(M)

-14(M)

-15(M)

-10(M)

-8(M)-17(D)

-19(D)

-11(M)

-21(D)

-7(M)-6(M)

-9(M)

-8(M)

-7(M)

-6(M)-10(M)-11(M)

m/z

α-synuclein

-9 -8 -7

500 1300700 900 1100arrival time (µs)

90 V

40 V

20 V

500 1300700 900 1100 arrival time (µs)

500 1300700 900 1100arrival time (µs)

(a) (b) (c)

α-synuclein

Open Open Open

Compact Compact Open

α-synuclein Summary

• Dimers form under specific solution condition

• Gas phase structures mimic solution structures

• Compact soln. structures open up with injection energy and charge

α-synucleinNMR and X-ray diffraction ineffectictive

Undergoes environmentally induced conformational changes• α-helical in Acidic Phospholipid Vesicles• Exhibits β-structure prior to fibril formation

Rg for small angle X-ray scatteringpH 7: Globular (15 Å) < 40Å α-syn < random coil (52 Å)Rg reduces in value 33% from pH 7 → pH 3

Conclusions

• Small Systems– Rearrange on Desolvation

• Large Systems– Retain Solution Structures Small Systems– Rearrange on Desolvation

• Intermediate Systems– Case depends on solvent stabilization

• Less in nucleotides• More in peptides/proteins

– Depends on Rearrangement Barriers

AcknowledgementsErin BakerDr. Jennifer GiddenAlexandra Fezuco

Erin BakerSummer Bernstein

Summer BernsteinDr. Thomas WyttenbachDr. Andrij BaumketnerDr. Joan Shea

AT

Thomas

Summer

Erin

AlyAly

JenJen