Relaxation compensation in chemical exchange measurements for the quantitation of amide hydrogen exchange in larger proteins

Abstract

AbstractThe increasingly rapid transverse relaxation of larger macromolecules serves to limit the practical length of various types of mixing periods. When chemical exchange dynamics are used to determine the rates of amide hydrogen exchange with the bulk solvent, the foreshortened mixing period results in lowered sensitivity. Three approaches are examined for increasing the practical length of the mixing period. The transverse relaxation rate of the amide resonances is decreased by perdeuteration of the carbon‐bound hydrogen positions and also by introduction of a TROSY‐based 15N‐separated pulse sequence. Reference experiments are proposed which provide accurate compensation for relaxation effects so that exchange rate data can be obtained over the entire mixing period profile. As a result, more than a 100‐fold range of amide exchange rates can be accurately determined for a moderate‐sized protein. Copyright © 2003 John Wiley & Sons, Ltd.