Abstract
In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.
Highlights
Hydrogen exchange provides important information on the structure and dynamics of proteins
Amide protons are locked into hydrogen bonds and may be solvent-obstructed, which makes hydrogen exchange significantly slower in comparison to amide sites located in unstructured, solvent-exposed regions
In order to quantify hydrogen exchange in a solid protein sample at equilibrium conditions, we followed the Relax-EXSY concept previously introduced in liquid state NMR by Lippens and coworkers [15]
Summary
Hydrogen exchange provides important information on the structure and dynamics of proteins. Amide protons are locked into hydrogen bonds and may be solvent-obstructed, which makes hydrogen exchange significantly slower in comparison to amide sites located in unstructured, solvent-exposed regions. Local and/or cooperative protein motion can open hydrogen bonds, and protons can be exchanged. Exchange rates in the timeframe of minutes to months can be obtained by monitoring the amide proton signal decay of lyophilized protein dissolved in D2O buffer [3]. Modifications to this approach allow the monitoring of faster exchange processes [4,5]
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