Mechanistic investigation of coordinated conformational changes in multisubunit ion channels and enzymes

Abstract

Many enzymes and ion channels consist of multiple subunits and/or multiple distinct functional components. Coordinated conformational changes through allosteric interactions between subunits and/or between functional units can efficiently regulate protein activity. This dissertation describes investigations of coordinated conformational changes in two systems: the ATP-sensitive potassium (KATP) channel and the ATP-dependent bacterial protease, ClpAP. KATP channels consist of two protein subunits: a pore-forming subunit, Kir6.2 and a regulatory subunit, SUR1. Kir6.2 is an inwardly rectifying potassium channel, and SUR1 belongs to the ATP-binding cassette (ABC) superfamily. Using patch clamp techniques, KATP channel activity was observed directly with single-channel resolution. The results indicate that noise from stochastic channel gating is significantly reduced compared to what would be observed for identical and independent channels, and provide evidence that negatively cooperative interactions between neighboring KATP channels are the source of the noise reduction. Simulations further suggest that negative coupling among KATP channels in pancreatic beta cells could be important for reliable signal transduction. Energetic coupling between Kir6.2 and SUR1 subunits was also investigated. Single-channel records were analyzed to detect the violations of microscopic reversibility in channel gating that would occur if Kir6.2 conformational transitions were driven by the energy from ATP hydrolysis by SUR1. Although no violations of detailed balance in channel gating are detected on the time scale where ATP hydrolysis takes place, unexpected non-equilibrium gating is observed on longer time scales. These results imply that channel gating is coupled to non-equilibrium processes other than ATP hydrolysis by SUR1. The second system studied for coordinated conformational change was ClpAP.

(cont.) ClpAP is composed of an ATPase, ClpA and a serine peptidase, ClpP. ClpA uses the free energy of ATP hydrolysis to unfold protein substrates and translocate them to ClpP, which proteolyzes them. To investigate how protein translocation by ClpA is coupled to proteolysis by ClpP, size distributions of peptide products were measured. The observation of non-exponential size distributions, in combination with simulations predicting how different mechanisms would influence the size distribution, supports the hypothesis that peptide product sizes are controlled by coordinated conformational changes of ClpA and ClpP.