Multi-Field CEST NMR Reconstructs a Protein Folding Landscape

Protein dynamics are central to their folding and function, yet the sparsely populated conformational states that underpin these processes remain difficult to characterize at atomic resolution. Chemical exchange saturation transfer (CEST) NMR can detect such states and is particularly sensitive to rapid exchange between two minor conformations. This "minor-state exchange", however, broadens and displaces the minor-state dip in CEST profiles, leading to parameter degeneracy that can preclude quantitative reconstruction of multistate exchange networks. Here, we show that, in the presence of rapid "minor-exchange", the apparent minor-state chemical shift obtained from two-state fits varies systematically with the static magnetic field, B0. We exploit this field dependence to develop a nonperturbative, multi-field CEST strategy that distinguishes otherwise degenerate exchange models. Applying multi-field 15N-CEST to the model protein Im7 revealed a folding intermediate populated at ∼1% that exchanges with the folded state on a ∼2 ms time scale and with an unfolded state populated at ∼0.4% on a ∼200 μs time scale. Although the unfolded state does not produce a separate CEST dip, its exchange is encoded in the line width and B0-dependent position of the intermediate-state dip. Analysis of the 15N-CEST data provides populations, microscopic exchange rates, and residue-specific 15N chemical shifts for all three states. The resulting Im7 free-energy landscape is sequential, with folding proceeding through the intermediate and no detectable direct exchange between the folded and unfolded states. Multi-field CEST provides a broadly applicable, perturbation-free approach for resolving biomolecular dynamics at equilibrium.