Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale

Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a ‘visible’ major conformer exchanges with two ‘invisible’ minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate—fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide ¹⁵N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although ¹⁵N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, ¹⁵N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of ¹⁵N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale (\({k}_{ex,FI1}\) ~ 550 s⁻¹, \({k}_{ex,FI2}\) ~ 1200 s⁻¹, \({k}_{ex,I1I2}\) ~ 5000 s⁻¹), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15 °C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.