Zagotta, William N.Eggan, Pierce2025-05-122025-05-122025-05-122025Eggan_washington_0250E_27880.pdfhttps://hdl.handle.net/1773/53017Thesis (Ph.D.)--University of Washington, 2025The cyclic nucleotide binding domain (CNBD) channels are an important family of ion channels that regulate various physiological roles including sensory perception, cardiac activity, and neuronal cellular excitability. These channels are regulated by the binding of cyclic nucleotides, which causes conformational changes throughout the protein structure that ultimately open the channel's pore. However, the details of this allosteric process, particularly the heterogeneity of conformational states and the energetic coupling between domains, remain poorly understood. This body of work utilizes time-resolved transition metal ion resonance energy transfer (tmFRET) techniques to investigate the conformational changes and energetics underlying ligand binding in a prokaryotic CNBD channel SthK. Time-resolved tmFRET involves the measurement of fluorescence lifetimes to determine the distance between a donor fluorophore and metal ion acceptor, enabling the detection of a protein's structural dynamics. The methodology was first validated here using the model protein maltose-binding protein (MBP) and then applied to SthK in first a C-terminal fragment (SthKCterm) and subsequently a full-length form (SthKFull). In SthKCterm we have been able to make measurements of intramolecular distance distributions within SthK's CNBD to monitor the conformational changes of this domain when ligand binds. These probability distributions were used to calculate the changes in Gibbs free energy (G) for both full agonist cAMP and partial agonist cGMP, showing that cAMP drives the conformational transition more than cGMP. In measurements of SthKFull, we have been able to compare distance distributions to those made in SthKCterm and revealed the influence of interdomain coupling, with the presence of the pore domain enhancing the CNBD's conformational transition. This work highlights the use of time-resolved tmFRET for characterizing the conformational dynamics and energetics of allosteric proteins in general and provides valuable insights into the ligand-mediated allosteric mechanism of SthK specifically.application/pdfen-USCC BYallosteryCNG channelconformational dynamicsFRETBiophysicsBiochemistryPhysiologyMolecular and cellular biologyThesis