The Structure of Small ProtonatedPeptides Containing Arginineand the Effect of Hydration

Thomas Wyttenbach, Denfeng Liu,Perdita Barran, Michael T. Bowers

University of California, Santa Barbara

Bela PaizsGerman Cancer Research Center,

Heidelberg, Germany

Vicki Wysocki, Linda BreciUniversity of Arizona, Tucson, AZ

Background� Building accurate computer models for protein

identification from MS/MS data requires knowledge of fragmentation mechanisms.

� Is the structure/conformation related to fragmentation pattern/mechanism?

� Does H/D exchange give information on peptide structure/conformation?

� Can H/D exchange and ion mobility data be structurally correlated?

Approach� Look at several series of peptides where important

groups are systematically varied.� Obtain

° H/D exchange° ion mobility° hydration energy° detailed ab initio/DFT data.

� Example series: RAAAA, AARAA, AAAAR� This talk: AARAA.

AARAA

2NNH

NHNH

NHO

O

O

O

O

OH

D vs Ldifferentresidue

O H2NNH

NHNH

NHO

O

O

O

O

OH

NH

H2N NH2+

NH

H3N+

zwitterion / salt bridge

O

O�

AARAA

2NNH

NHNH

NHO

O

O

O

O

OH

D vs Ldifferentresidue

OO H2NNH

NHNH

NHO

O

O

O

O

OH

NH

H2N NH2+

NH

NH

OO

blocked termini

H/D exchange in the Ion Trap

MH+

H/D

430 432 434 436 438 440 4420

25

50

75

100

Rel

ativ

e In

tens

ity

m/z

MH+ MD+

458 460 462 464 466 468 4700

25

50

75

100

500 502 504 506 508 510 5120

25

50

75

100

Rel

ativ

e In

tens

ity

472 474 476 478 480 482 4840

25

50

75

100

m/z

AARAA

MH+

MH+ MH+

AARAA-O-CH3 acetyl-AARAA

H2NNH

NH

O

O

NH

O

NHOH

O

O

HN

H2N NH

H

3 fast exchanges

AARAA:AARAA-OMe:

Ac-AARAA:

H/D exchangeNo H/D exchangeNo H/D exchange

Both termini involved in (AARAA)H+ H/D exchange

Is (AARAA)H+ a salt bridge???

Relay Mechanism forH/D exchange with D2O

NHNH

ONH

ONH

NHOH

O

O

O

O

Campbell, Rodgers, Marzluff, Beauchamp,

JACS, 1995, 12840-12854

H+

OD

D

NHNH

ONH

ONH

NHOH

O

O

O

O

Campbell, Rodgers, Marzluff, Beauchamp,

JACS, 1995, 12840-12854

H OD

D+

Birthday!!!Happy

Campbell, Rodgers, Marzluff, Beauchamp,

JACS, 1995, 12840-12854

Relay Mechanism forH/D exchange with D2O

Relay mechanisminvolving salt bridge

OD

DN

O�

H OH

H

Ion mobility method

ESI IonSource

ESI IonSource

IonFunnel

IonFunnel

DriftCell

DriftCell MSMS DetectorDetector

in out

E

Drift cell1�5 torr He

DriftCell

DriftCell

Cro

ss S

ectio

n (Å

2 )

130

135

140

145

150

155

160

165

170

�0 �1 �2 �3

AA

RA

A

AA

RA

A-O

Me

Ac-

AA

RA

A

Experimental cross sections

Experiment: drift time ion mobility experimentalcross section

Calculation: model structures calculatedcross section

Molecularmodeling:

AMBER

(AARAA)H+

Lowest energy structure (AMBER)

Charge solvation Zwitterion (salt bridge)

+ +

+

�

open compact

Cro

ss S

ectio

n (Å

2 )

Experiment

Calculation: Charge Solvation

Calculation: Salt Bridge

AA

RA

A

AA

RA

A-O

Me

Ac-

AA

RA

A

130

135

140

145

150

155

160

165

170 Cross sections

H/D-exchange ( ): salt bridge (?)(AARAA)H+ + H2O

Ion mobility ( ): no salt bridge(AARAA)H+

H2O

Calculations

MM scan / DFT optimizationB3LYP/6-31+G(d,p)

10.9

4.8

0.0

(AARAA)H+

Energy (kcal/mol)

Zwitterion (>C=OH+)

Zwitterion (�NH3+)

Charge solvation

Structure(AARAA)H+

+ H2O

�

1.8

0.0

0.0

(AARAA)H+aq

Charge solvation

Salt bridge

(AARAA)H+

Experiment:σ = 145 Å2

σ = 151 Å2

σ = 137 Å2

(AARAA)H+�H2Oglobal minimum

(charge solvation)

H2O

Zwitterion (+5 kcal/mol)

C-terminus

N-terminus

set up forH/D exchange

relay mechanism

(AARAA)H+�H2O

C-terminus

N-terminus

Transition state (AARAA)H+�H2O

C-terminus

N-terminus

Charge solvation (AARAA)H+�H2O

C-terminus

N-terminus

Transition state (AARAA)H+�H2O

Zwitterion

C-terminus

N-terminus

(AARAA)H+�H2O

458 460 462 464 466 468 4700

25

50

75

100

500 502 504 506 508 510 5120

25

50

75

100

Rel

ativ

e In

tens

ity

472 474 476 478 480 482 4840

25

50

75

100

m/z

AARAA

MH+

MH+ MH+

AARAA-O-CH3 acetyl-AARAA

H2NNH

NH

O

O

NH

O

NHOH

O

O

HN

H2N NH

H

3 fast exchanges

(AARAA)H+ + D2O (AARAA)D+ + HOD

2 kcal/mol

11 kcal/mol

ZWZW

CS

(AARAA)H+ ··· D2O

CSTS

Relay mechanism

B3LYP/6-31+G(d,p)

(AARAA)H+ + D2O (AARAA)D+ + HOD

2 kcal/mol

11 kcal/mol

ZWZW

CS

(AARAA)H+ ··· D2O

CSTS

Relay mechanism5 kcal/mol

B3LYP/6-31+G(d,p)

Ion mobility instrumentation:Hydration under equilibrium conditions

ESI IonSource

ESI IonSource

IonFunnel

IonFunnel

DriftCell

DriftCell MSMS DetectorDetector

in out

E

Drift cell1�2 torr H2O

DriftCell

DriftCell

M+ M+�(H2O)n

450 475 500 525 550 575 600

Mass spectrum 1.3 torr H2O

260 K

m/z

AARAA 18

3

MH+ + n H2O MH+�(H2O)n

1 45

2

=0n

450 475 500 525 550 575 600

450 475 500 525 550 575 600

3

MH+ + n H2O MH+�(H2O)n

1.3 torr H2O260 K

1 45

2

=0n

m/z

b) AARAA-OMe

a) AARAA

Mass spectra

2118

9.48.4

12

AARAA-OMe

2118

9.58.1

12

Ac-AARAA

23 ± 118

10.2 ± 0.38.4

12

AARAA

�∆∆∆∆Sº(cal/mol/K)

�∆∆∆∆Hº(kcal/mol)

nM

Experimental ∆∆∆∆Hº and ∆∆∆∆Sº valuesMH+�(H2O)n�1 + H2O MH+�(H2O)n

Water binding energy(AARAA)H+ ··· H2O

Theory ( ): 8.9 kcal/mol ( )Experiment ( ): 10.2 kcal/mol

after BSSEcorrectionand ZPE

H/D-exchange: (AARAA)H+ salt bridge?

Ion mobility: (AARAA)H+ no salt bridge

Theory: � no salt bridge for (AARAA)H+

� low TS for H/D-exchange for all 3 N-terminus hydrogens from salt bridge form

� no exchange possible for blocked termini since TS for proton transfer to >C=O groups high in energy

� H2O stabilizes (AARAA)H+ salt bridge more than charge solvation

Summary