Abstract
How can a single protein domain encode a conformational landscape with multiple stably folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from
. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) states. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single-sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the
isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase Cyclophilin A. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We found two additional properties of KaiB's landscape. First, the Ground state experiences cold denaturation: At 4 °C, the PD state becomes the majorly populated state. Second, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond-timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this Enigma state is a beta-strand register shift that relieves buried charged residues, and support this structure experimentally. These results provide mechanistic insight into how evolution can design a single-sequence that achieves specific timing needed for its function.