UC Merced

Circadian clocks are universal among living organisms. In humans, circadian clock serve two main function; (1) promote daytime responses such as physical and mental activity and the biochemical and cardiovascular changes associated with them and (2) to enable preparation between the active and resting phases. To better understand how circadian clocks exert these functions it is necessary to study the molecular mechanism governing them. The circadian clock of cyanobacteria is a good model for studying such mechanisms due to its simplicity and ability to be reconstituted in vitro. Of the three core oscillating proteins, KaiA, KaiB, and KaiC, KaiB is a unique metamorphic protein that undergoes a fold-switching mechanism allowing KaiB to sample two native folds. Comparison of these folds revealed three prolyl trans-to-cis isomerase residues. To elucidate the mechanism behind KaiB fold switching, mutagenesis of the three proline residues to alanine was applied. It is hypothesized that removing the prolyl trans-cis isomerization will cause KaiB to become more stable by restricting fold switching. Differential scanning calorimetry of the proline to alanine mutants showed destabilization of proline70 to alanine suggesting Pro70 may act as a molecular hinge. NMR relaxation experiments were optimized for future studies on the destabilizing effect of Pro70 to alanine and its role as a potential molecular hinge.