Increasing the accuracy of exchange parameters reporting on slow dynamics by performing CEST experiments with highB1fields

Abstract

AbstractOver the last decade chemical exchange saturation transfer (CEST) NMR methods have emerged as powerful tools to characterize biomolecular conformational dynamics occurring between a visible major state and ‘invisible’ minor states. The ability of the CEST experiment to detect these minor states, and provide precise exchange parameters, hinges on using appropriateB1field strengths during the saturation period. Typically, a pair ofB1fields withω1(= 2πB1) values around the exchange ratekexare chosen. Here we show that the transverse relaxation rate of the minor state resonance (R2,B) also plays a crucial role in determining theB1fields that lead to the most informative datasets. Using, to guide the choice ofB1, instead ofkex, leads to data wherefrom substantially more accurate exchange parameters can be derived. The need for higherB1fields, guided by K, is demonstrated by studying the conformational exchange in two mutants of the 71 residue FF domain withkex∼11 s-1and ∼72 s-1, respectively. In both cases analysis of CEST datasets recorded usingB1field values guided bykexlead to imprecise exchange parameters, whereas usingB1values guided by K resulted in precise site-specific exchange parameters. The conclusions presented here will be valuable while using CEST to study slow processes at sites with large intrinsic relaxation rates, including carbonyl sites in small to medium sized proteins, amide15N sites in large proteins and when the minor state dips are broadened due to exchange among the minor states.