Abstract
Circadian rhythm plays an essential role in many biological processes. In cyanobacteria, circadian rhythm is regulated by three core proteins: KaiA, B, and C.1 KaiB is an example of a metamorphic protein, a protein that can adopt more than one conformation. In some systems, the circadian rhythm is regulated by KaiB and C, with KaiB binding to KaiC only in the fold-switch (FS) state.1 When not bound to KaiC, it exists in a ground state.1 It is estimated that roughly 0.5-4% of all proteins in the PDB are fold-switching proteins.2 Multiple sequence alignment (MSA) of known protein states can be used to predict the less likely state of metamorphic proteins using AlphaFold-2 (AF2).4,29 AF2 has become the most accurate tool to predict protein conformations but has lacked in predicting multiple conformations of metamorphic proteins.3 The Kern lab has shown that AlphaFold-cluster (AF-cluster), which utilizes MSA, can predict both states of KaiB.4 It was also used to find natural variants of KaiB that exist in the FS state. One such KaiB was that of T. elongatus vestitus (KaiBTV).4 In order to verify the FS state was the most populated conformation, I expressed and purified KaiBTV for verification of conformation using nuclear magnetic resonance (NMR).Casein Kinase I (CK1 is a serine/threonine kinase that maintains the core clock of the human circadian rhythm by phosphorylating its protein substrate, human Period protein 2 (hPER2).6-16 While most kinases are known for having fast activity, CK1phosphorylates hPER2 slowly in order to maintain the 24-hour circadian period.6-22 Phosphorylation of hPER2 allows it to be transported into the nucleus, along with Cryptochrome (CRY) and CK1, to regulate transcription of hPER2.6-16 While it is known that the Familial Advanced Sleep Phase (FASP) region interacts directly with the kinase, other interactions that might affect the affinity of hPER2 to the kinase are not fully understood. Understanding the mechanism of the interaction between CK1 with hPER2 would demonstrate how the two dictate the 24-hour circadian rhythm. There are many CK1 mutations defined in the literature that either shorten or lengthen the period but the mechanism by which some of these mutants alter the period is somewhat unknown.7,11,16,22 Period shortening mutations are found to be conserved in both CK1 and DBT, the Drosophila homolog of CK1.7,11,16,22 Seeing how these mutations affect affinity and phosphorylation kinetics would give information on how CK1 and hPER2 maintains the 24-hour period. Preliminary activity assay data suggests that the landing site of hPER2 leads to higher phosphorylation activity by CK1C, but more work needs to be done to verify that the landing site is crucial for phosphorylation of the FASP region.