UCLA

Proteins in solution are dynamic molecules that exhibit conformational flexibility across a range of time and length scales, and characterizing the functional role of protein motion is a primary goal in molecular biophysics. High hydrostatic pressure has emerged as a powerful probe of protein conformational flexibility. Development of instrumentation and methodologies that enable electron paramagnetic resonance (EPR) experiments on proteins at high pressure is the central aim of the work presented in this dissertation.

Pressurization of proteins reveals regions of elevated compressibility, and thus flexibility, within individual conformational states, but also shifts conformational equilibria such that “invisible” excited states become accessible for spectroscopic characterization. Current evidence indicates that pressure cleanly shifts the relative populations of states solely according to differences in partial molar volume without altering the shape of the conformational free energy landscape. Thus, variable pressure is a powerful tool for dissecting details of the landscape, and site-directed spin labeling coupled with electron paramagnetic resonance spectroscopy (SDSL EPR) is an ideal strategy in terms of sensitivity and time scale to detect the effects of pressure and interpret them in terms of structure and dynamics. In this dissertation, newly developed high-pressure instrumentation for both variable-pressure continuous-wave EPR and pressure-resolve double electron-electron resonance (PR DEER) of proteins in aqueous solution is described. The applications presented illustrate the considerable potential of the methods to: (1) identify compressible (flexible) regions in a folded protein; (2) determine thermodynamic parameters that relate conformational states in equilibrium; (3) populate and characterize excited states of proteins undetected at atmospheric pressure; (4) reveal the structural heterogeneity of conformational ensembles and provide distance constraints on the global structure of pressure-populated states. The SDSL EPR results are complemented by global secondary structure information provided by high-pressure circular dichroism experiments.

This work lays the foundation for future developments in high-pressure SDSL EPR, including pressure-jump relaxation spectroscopy to determine the lifetime of conformational states in the millisecond range and high pressure saturation recovery exchange spectroscopy to enable measurement of lifetimes of states in the microsecond range. SDSL EPR has unique advantages for the study of membrane proteins in their native environment under physiological conditions, and applications of high-pressure SDSL EPR to explore the conformational equilibria and dynamics of integral membrane proteins is a high priority for future work.