Folding Thermodynamics and Kinetics of the N-Terminal Domain of the Circadian Clock-Regulated Histidine Kinase SasA.

Despite groundbreaking advancements in protein structure prediction, particularly with AlphaFold2/3 and RoseTTAFold, the protein folding problem remains elusive. In this study, we investigate the folding kinetics of the N-terminal thioredoxin-like domain of histidine kinase SasA (N-SasA), a key component of the two-component regulatory system of the cyanobacterial circadian clock, and its dependence on proline cis/trans isomerization. Using a multidisciplinary approach, we evaluated the thermodynamic stability of N-SasA under thermal and chemical denaturation conditions and determined the energy barriers for folding and unfolding. Intrinsic tryptophan fluorescence and nuclear magnetic resonance spectroscopy revealed that proline isomerization is a rate-limiting step in folding, as evidenced by accelerated kinetics in the presence of peptidyl-prolyl cis/trans isomerase.