Salt Bridges: Aggregation, Hydration, and Fragmentation of Peptides and ligonucleotides UCSB: Thomas Wyttenbach, Perdita Barran, Jennifer Gidden, Summer Bernstein, Dengfeng Liu, Mike Bowers U. Arizona: Linda Breci, Vicki Wysocki He idelberg: Be la P a izs $$ NSF (UCSB)
Structure of small peptides H/D exchange vs. ion mobility Aggregation of peptides Salt bridges in oligonucleotides?
Building accurate computer models for protein identification from MS/MS data requires knowledge of fragmentation mechanisms. Is the structure/conformation related to fragmentation pattern/mechanism? Does H/D exchange give information on peptide structure/conformation? Can H/D exchange and ion mobility data be structurally correlated?
Look at several series of peptides where important groups are systematically varied. Do H/D exchange and ion mobility studies on each group. Measure hydration energies. Do detailed ab initio/dft calculations on selected systems. Here we will discuss RAAAA, AARAA, AAAAR but focus on AARAA.
University of Arizona, Tucson University of California, Santa Barbara German Cancer Research Center Heidelberg
2N H 2 N NH different residue NH NH + H 2 N NH 2 NH NH NH NH NH DvsL NH NH H H NH
MH + MD + Relative Intensity 100 75 50 25 MH + H/D 0 430 432 434 436 438 440 442 m/z
100 75 50 MH + 3 fast exchanges AARAA H 2 N HN H N H Relative Intensity 25 0 100 NH H 2 N 458 460 462 464 466 468 470 MH + MH + 100 NH NH NH H 75 AARAA--CH 3 75 acetyl-aaraa 50 50 25 25 0 472 474 476 478 480 482 484 0 500 502 504 506 508 510 512 m/z
' 25HD\0HFKDQLVPIRU+'H[FKDQJH NH H NH NH D D NH H D NH D NH H &DPSEH5RGJHUV0DU]XII%HDXFKDPS -$&6
AARAA AARAA-Me Ac-AARAA Exchange YES N N %RWKWHUPLQLLQYRYHG 6DWEULGJH"
>$$5$$@+ /RZHVWHQHUJ\VWUXFWXUH$0%(5 + + + &KDUJHVRYDWLRQ =ZLWWHULRQ VDWEULGJH
Ion mobility method (UCSB) Ion Source MS1 Drift Cell MS2 Detector
Ion mobility method in out
,RQ0RELLW\8&6% Experiment Calculated (zwitterion) Calculated (charge solvation) [Ac-AARAA]H+ [AARAA]H+ 132 134 136 138 140 142 144 146 148 150 152 154 156 158 Cross section (Å 2 )
Peptide σ (Experiment) σ (AMBER Calculation) Neutral Terminus Zwitterions Terminus [RAAAA]H + 136.43 139.38 136.38 [AARAA]H + 145.11 146.52 136.40 [AAAAR]H + 147.51 148.77 139.79 [AcRAAAA]H + 144.16 144.25 -------- [AcAARAA]H + 151.38 148.29 -------- [AcAAAAR]H + 151.84 151.64 -------- ( )
(AARAA)H + + H 2 (AARAA)H +
(Heidelberg) B3LYP/6-31g(d)
Structure Energy (kcal/mol) (AARAA)H + (AARAA)H + + H 2 Charge solvation Zwitterion ( NH 3+ ) Zwitterion (>C=H + ) 0.0 2.3 12.4 0.3 0.0
(AARAA)H + + H 2 Zwitterion setup for relay mechanism: 14.1 kcal/mol + C-terminus + N-terminus H 2
(AARAA)H + + D 2 (AARAA)D + + HD 7 kcal/mol 16 kcal/mol Relay ZW Relay TS Relay CS ZW 2 kcal/mol 5 kcal/mol CS (AARAA)H + D 2
(AARAA)H + H 2 Theory (Heidelberg): 16 kcal/mol Experiment (UCSB): 10 kcal/mol (BSSE correction? Larger basis set?)
H/D-exchange: salt bridge (?) (AARAA)H + H 2 Ion mobility: no salt bridge (AARAA)H + Theory: no salt bridge for (AARAA)H + salt bridge for (AARAA)H + H 2 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= groups high in energy
Important in certain diseases -Alzheimers -Mad Cow (TSE) - Diabetes type II Important for chaperone formation bserved as (common) occurrence in electrospray of peptides Energetics and mechanism not yet known
Beauchamp et al.: penta-, hexa-peptides Clemmer et al.: BK, Insulin chain A, ala 12 Jarrold et al.: ac-lys-ala 19, ac-(gly-ala) 7 -lys Bowers et al.: BK, LHRH, neurotensin But little detailed understanding of factors that control this aggregation
Bradykinin 9 residue peptide 2 Arg 1 Acidic functional group Zwitterion formation H2 N PR3 PR2 C N ARG1 CH C N C GLY4 H N CH C H N H PHE5 CH C CH 2 SER6 H N CH C CH 2 H PR7 PHE8 ARG9 N C H N H 2 C CH C H N H 2 C CH C CH 2 CH 2 HN C NH 2 NH H CH 2 CH 2 CH 2 NH PGLU 1 1 2 + 1 2 & &+ & + & & +1 + 1 C NH PR9 NH 2 2 + HIS2 TRP3 SER4 TYR5 GLY6 LEU7 ARG8 & 1 2 2 2 2 2 2 2 + + + + + &+ & 1 &+ & 1 &+ & 1 &+ & 1 &+ & 1 &+ & 1 &+ & 1 + &+ &+ &+ &+ + &+ &+ 1+ +1 2+ 2+ &+ &+ &+ &+ + & 1+ & 1+ 1+ GLY10 2 &+ & 1+ + LHRH 10 residue peptide 1 Arg no acidic group Blocked Termini
Ion Arrival Time Distribution at m/z = 354 Ion Arrival Time Distribution at m/z = 531 Ion Arrival Time Distribution at m/z = 1061 Bradykinin Time Time Time (M+2H) 2+ (M+3H) 3+ (M+H) + 200 300 400 500 600 700 800 900 1000 1100 1200 m/z Mass Spectrum
Injection Voltage 105 V 75 V Conclude: A = (3BK+3H) 3+ B = (2BK+2H) 2+ C = (BK+H) + 60 V 45 V B A C Arrival time
Increased Cell Temperature (2M+2H) 2+ 2(M+H) + dissociation Fitting the ATDs yield rate constants Binding energies (E a ) are obtained from an Arrhenius type of analysis
[2BK+2H] 2+ 2[BK+H] + 503 K [BK+M] + 460 K 2[BK+2M] 2+ Arrival time 310 K
Fit ---- Data 500 600 700 800 900 time (µs)
2+ + ln(k) 10 9.5 9 8.5 8 7.5 7 6.5 6 5.5 5 Bradykinin E a = 35.1±1 Kcal/mol A = 5.1 *10 19 s -1 S = 28.8 cal mol -1 K -1 0.002 0.00205 0.0021 0.00215 0.0022 0.00225 0.0023 1/T (K -1 )
LHRH [M+H] 2+ [M+H] + Arrival Time 400 600 800 1000 1200 m/z
2+ + ln(k) 10 9.5 9 8.5 8 7.5 7 6.5 6 5.5 490 K Bradykinin E a = 35.1 ± 1 Kcal/mol S = 28.8 cal mol -1 K -1 456 K LHRH E a = 51.7 ± 2 Kcal/mol S = 69.0 cal mol -1 K -1 445 K 435 K 5 0.002 0.00205 0.0021 0.00215 0.0022 0.00225 0.0023 0.00235 1/T (K -1 )
Simulated annealing Dynamics simulations (300 K)
(2M+2H) 2+ (TS) 2(M+H) + Bradykinin (TS) + LHRH (TS) +
9.8 Å Monomers only slightly rearranged in dimer Electrostatic binding between charged groups dominates 9.9 Å 7.2 Å 9.1 Å 12.7 Å
3.7 Å N C 6.0 Å N C 13.4 Å Intermolecular interactions in dimer force open the backbone of the monomer Causes repulsion of the 2 N-termini and attraction between opposing N and C termini N 9.6 Å C
Energy [2M+2H] 2+ E a = 35.1 kcal/mol (exp) E a = 51.7 kcal/mol (exp) 2[BK+H] + 24.5 kcal/mol (AMBR) 2[LHRH+H] + 11.2 kcal/mol (AMBER) Rxn Coordinate
Do our calculated structures agree with experiment? Compare Cross Sections (Å 2 ) Experiment Theory* σ (%) [LHRH+H] + 257 261 +1.5 [2LHRH+2H] 2+ 409 417 +1.9 [BK+H] + 240 242 +0.8 [2BK+2H] 2+ 384 376 2.1 * minimized structures (lowest 10 kcal/mol)
Peptides tend to form aggregates of the form (zm+zh) z+. Energy barriers for dimer dissociation are of the order of 30-50 kcal/mol both for zwitterion (BK) and charge solvation structures (LHRH). Entropies (and energies) of activation indicate a major structural change occurs in TS for LHRH (i.e. a loose TS) while the TS for BK is much more dimer like (i.e. tighter).
Mass spe ctromet ric anal ysis of DNA crucial for obt a ining rapi d informat ion o n sma ll s amp les Problem: DNA fragment s du ring the sa mpling p roces s Solution: Unde rs tand th e fra gmentat ion me chan ism Propo sed mecha nis m: 1. proton ation of bas e 2. wea ken / b rea k ba se suga r bond 3. eliminate b ase a nd induce bac kbone fragmen tatio n
Proto n affinitie s of t he 4 DNA base s: G > A C >> T Hillenkam p, G ross: Look at f ragm e ntat ion of o ligonuc leot ides with T and G ba s es shoul d see pre fer e ntia l loss of G dgttt, dtgtt, dttgt, dtttg po s itive ion s : s e e lo s s of G in a ll case s but for negat ive ions
dtttg (dgttt )
ossible explanation: dtgtt and - dttgt form - salt bridges H CH 2 HN N CH 3 P - CH 2 + H N N N NH NH 2 P - HN N CH 3 CH 2 P H CH 2 HN N CH 3 H J. Gross, F. Hillenkamp, K.X. Wan, M.L. Gross J. Am. Soc. Mass Spectrom. 2001, 12, 180
Look at a simpler model with same possibility of salt bridge formation Is dtgt - a salt bridge? H H N CH 3 CH 2 N P - CH 2 H + N N N NH NH 2 P - HN CH 3 N CH 2 H
ATDs for dtgt - 300 K 80 K σ = 17 Å 2 arrival time
Scatter Plot for dtgt (salt bridge) - Cross-Section ( 240 230 220 210 200 190 180 170 160 expt Å ) 2 0 5 10 15 20 25 30 35 40 Relative Energy (kcal/mol)
Scatter Plot for dtgt (singly deprotonated) - 250 240 230 Cross-Section (Å 220 210 200 open 2 ) expt 190 180 folded 0 5 10 15 20 25 30 Relative Energy (kcal/mol) 2 families of low-energy conformers "folded" smaller cross-section "open" larger cross-section σ ~ 18 Å 2 σ expt ~ 17 Å 2
Similar ATDs for dgtt -and dttg - (which should not be salt bridges) dgtt - dtgt - dttg - σ = 194 Å 2 σ = 196 Å 2 σ = 195 Å 2 300 K folded open folded open folded 80 K open arrival time arrival time arrival time
Con cl us ion s : dtgt - not a sa lt bridge dtgtt - an d dttgt - a lso not sa lt bridges Gros s / Hillen kamp me chan ism may not be correct (or sa lt bridge is type of tra ns ition stat e ) Prelimina ry calcula tions indicate salt b ridge s tructure is highe r in e ne rgy Hydrat ion Studie s b e ing initiated
Small peptides and oligonucleotides are generally not salt bridges. However, salt bridges can be energetically close to charge solvation structures. Salt bridges are stabilized (compared to CS) by additional solvation. Salt bridges are important intermediates (H/D exchange, fragmentation). Little structural change occurs within a salt bridge unit when units aggregate.