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)
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