Development of New Methods for Electric Field-Stimulated X-Ray Crystallography

Abstract

This dissertation focuses on the development and expansion of electric field-stimulated X-ray crystallography (EF-X) as a novel biophysical method. EF-X applies a strong electric field to a macromolecular crystal concurrent with X-ray irradiation. The electric field creates a pattern of forces on local charged groups, inducing motions that can be resolved from the diffraction data as molecular structural changes. A 2016 EF-X proof-of-concept study demonstrated the promise of this method to provide both mechanistic and thermodynamic information on biological systems; nonetheless, major practical limitations persisted. In Chapter 2, I describe novel hardware and experimental protocols that address these limitations. I develop custom-designed microelectrodes that can deliver an electric field without the physical strain, dehydration, and osmotic shock that had plagued the earlier electrodes. I show that the combination of these novel microelectrodes with more robust sample mounting and data acquisition dramatically improves EF-X sample success rates and widens the range of compatible protein crystals, transforming the method into a fully-realized technology. In Chapter 3, I present EF-X data on two model systems facilitated by these electrodes. I demonstrate that the resulting EF-X data can yield key biochemical insights into phenomena such as ion conduction, but that there is more work to be done in refining techniques for EF-X data analysis. Next, I expand EF-X beyond the need for a polychromatic (Laue) X-ray source by combining the method with Hadamard time-resolved X-ray crystallography (HATRX). HATRX experiments sum together multiple time points on a single diffraction image, then repeat the measurement while varying the time point sequence. The resulting images can be iii deconvolved to generate diffraction data at individual timepoints. In Chapter 4, I describe a novel implementation of EF-HATRX, culminating in successfully collecting the first-ever EF-HATRX data. Finally, in Chapter 5 I assess different methods for deconvolving HATRX data more generally and show that the EF-HATRX data yield robust electron density from even the shortest, 500 ns timepoint.