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.