amide proton exchange nh + h* -> nh* + h forms of h – 1 h, 2 h, 3 h, 1 h sat amide nh usually...

Amide Proton Exchange

NH + H* -> NH* + H

Forms of H– 1H, 2H, 3H, 1Hsat

Amide NH usually measured– Thiol SH, Hydroxyl/Carboxyl OH, Amine NH3

+

– CH do not exchange (except special His ring CH)

Information Obtained by Amide Proton Exchange

NH + H* -> NH* + HHydrogen BondsLigand-binding SitesLigand-induced Conformational Changes

- Allosteric Changes

Conformational BreathingEnergy LandscapeIdentify Flexible Regions of ProteinsProtein Folding Mechanisms / Intermediates

Methods of MeasurementWith 3H: Freeze Drying; Dialysis; Gel Filtration; HPLCMass Spectrometry (bulk or fragments)Neutron Diffraction CrystallographyNuclear Magnetic Resonance Spectroscopy

– 1H / 2H exchange– Lineshape Analysis– Saturation Transfer

Infrared and IR Spectroscopy N-methylacetamide

N-H N-DAmide I (C=O stretch) 1653 cm -1 / 1642 cm-1

Amide II (N-H bend / C-N stretch) 1567 cm-1 / 1475 cm-1

Amide III (C-N stretch / N-H bend) 1299 cm-1 / 960 cm-1

Amide A N-H stretch (Fermi resonance) 3280 cm-1 / -Amide B N-H stretch (Fermi resonance) 3090 cm-1 / -

Amide Proton Exchange

Amide Proton ExchangeProtection FactorF = kex,bound / kex,free

SUPREXGhaemmaghami et al. PNAS 97: 8296 (2000)