Majumdar, Abir2021-08-162021-08-162021-05https://hdl.handle.net/11299/223147University of Minnesota Ph.D. dissertation. May 2021. Major: Pharmacology. Advisor: Nicholas Levinson. 1 computer file (PDF); xiv, 104 pages.Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. Using a series of DEER and NMR experiments, we studied the activation process of the G1/S kinase Cdk2 in solution. We show that catalytically inactive Cdk2 readily adopts multiple active-like states for efficient dephosphorylation, and that regulatory phosphorylation on the activation loop enhances allosteric coupling with the cyclin subunit. We then used DEER and FRET experiments to measure the binding of multiple CDK inhibitors and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. We reveal that the allosteric coupling between these biochemical effectors is responsible for the differential recognition of Cdk2 and Cdk4 inhibitors. Finally, we used sequence analysis, DEER, FRET and activity assays to identify and measure the effects of mutating an allosteric hub that has diverged between Cdk2 and Cdk4. We demonstrate that this hub controls the strength of allosteric coupling, and that the altered architecture and allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).enbiophysicschemical biologyconformationDEEREPRkinaseThesis or Dissertation