Title: Studies of Allostery in the Potassium Channel Kcsa by Solid-state NMR
Description: In this thesis, I focus on studies of the mechanism of inactivation in KcsA. Allosteric coupling between the pH gate and the selectivity filter in the protein is hypothesized to be the cause of inactivation. Allosteric coupling refers to changes at one site of a protein due to perturbations at a remote site. In chapter 3, I measured the potassium affinities at the selectivity filter at neutral and low pH, which corresponds to the closed and open conformation at the pH gate. The results show a three order of magnitude shift in the potassium affinity. This is direct evidence that the pH gate and the selectivity filter are coupled, in support of the activation-coupled inactivation hypothesis. The allosteric coupling factor, defined as the ratio of the affinities, can be used as a benchmark to study other factors in the allosteric process, such as the membrane and specific residues. Because of the potential deleterious effect of the acidic pH on the protein and membrane, we studied a mutant E118A&H25R, in which the pH gate is mutated to be open.

Thus we were able to measure the K+ affinity change in the open and closed conformation at the pH gate at neutral pH. The results confirmed that the opening of the pH gate results in an energetic stabilization of the collapsed (K+-unbound) state, and shifts the K+ affinity towards looser binding. In chapter 4, I tested the important role of residue F103 in mediating allosteric coupling, as suggested by electrophysiology and crystallography studies. I mutated this residue and measured the allosteric coupling factor on the mutant. The affinity at low pH is much tighter than wild-type and the coupling factor is significantly reduced. From the spectra, I observe local structural changes on I100 and T74 as a result of F103A mutation, implying the interaction among F103, I100 and T74 to mediate the allosteric coupling. F103 is distant from the pH gate and the selectivity filter; its effect on the coupling and inactivation behaviors confirms that inactivation involves coupling between the pH gate and the selectivity filter.

In chapter 5, I developed a method to probe those allosteric participants, such as F103 in KcsA by NMR measurements. I tested this method on KcsA, dissecting KcsA into various functional compartments. Various allosteric participants T75Cg T74Cg I100 were identified. The importance of residue T74 for the coupling was confirmed by electrophysiology and NMR thermodynamics characterization. In chapter 6, we applied SSNMR to probe the structural and magnetic properties of superatom clusters.