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Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two‐dimensional NMR
Author(s) -
Zhang YuZhu,
Roder Heinrich,
Paterson Yvonne
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040420
Subject(s) - chemistry , solvent , amide , catalysis , cyclohexane , proton nmr , quenching (fluorescence) , inorganic chemistry , organic chemistry , physics , quantum mechanics , fluorescence
In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein‐ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO‐D 2 O mixtures was investigated by NMR methods. As in water, H‐D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid‐ and base‐catalyzed processes and negligible water catalysis. However, the base‐catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is ∼ 100‐fold reduced relative to that in D 2 O. The solvent‐dependent decrease in base‐catalyzed exchange rates can be attributed primarily to a large increase in p K a values for the NH group, whereas solvent effects on p K w seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent‐quenching method, melittin was exchanged in D 2 O (pH 3.2, 12 °C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D 2 O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.