Increasing the accuracy of exchange parameters reporting on slow dynamics by performing CEST experiments with ‘high’ B1 fields

Over 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 appropriate B₁ field strengths during the saturation period. Typically, a pair of B₁ fields with ${\omega }_1$ (=${2\pi B}_1$) values around the exchange rate k_ex are chosen. Here we show that the transverse relaxation rate of the minor state resonance ($R_{2,B}$) also plays a crucial role in determining the B₁ fields that lead to the most informative datasets. Using $K={\left(k_{\mathrm{ex}}\left(k_{\mathrm{ex}}+R_{2,B}\right)\right)}^{\frac{1}{2}}$ ≥ k_ex, to guide the choice of B₁, instead of k_ex, leads to data wherefrom substantially more accurate exchange parameters can be derived. The need for higher B₁ fields, guided by $K$, is demonstrated by studying the conformational exchange in two mutants of the 71 residue FF domain with $k_{ex}$ ∼ 11 s⁻¹ and ∼ 72 s⁻¹, respectively. In both cases analysis of CEST datasets recorded using B₁ field values guided by $k_{ex}$ lead to imprecise exchange parameters, whereas using B₁ values 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, amide ¹⁵N sites in large proteins and when the minor state dips are broadened due to exchange among the minor states.