UCSF

Throughout history, methodological innovations have resulted in breakthroughs in our understanding of biology. Methods for determining static protein structures, as well as those for probing protein dynamics, are well-established. Nonetheless, visualizing molecules as dynamic entities that respond to their environment is still an outstanding challenge. Specifically, it is challenging to measure the spatial position of all the atoms within a molecule as a function of time. That challenge is the broad focus of this dissertation.

In chapter one, I begin by diving into modern crystallographic techniques that enable one to solve protein structures from sub-micron-sized crystals. I compare and contrast two methods, serial crystallography and electron crystallography, asking how each technique affects the protein’s structure. A primary factor differentiating these two methods is the temperature of the sample during the experiment. Despite this difference, both methods enable one to solve high-resolution structures from small crystals. This is advantageous for time-resolved experiments. Since there are fewer molecules in a small crystal, the perturbation is more uniform, which provides a clearer time-resolved signal.

In chapter two, I investigate temperature-jumps as a generalized perturbation for resolving the energy landscape of proteins. In this work, I focus on solution scattering experiments, which allow one to examine large-scale perturbations to a protein, as well as changes in the solvent shell surrounding the molecule. By mutating selected residues, we inhibited specific protein motions. Comparing these mutants to the wild-type protein allowed us to resolve the motions driven by an infrared laser. Nonetheless, we wished to gain all-atom spatial resolution, which required us to perform a temperature-jump within the context of crystallography rather than solution scattering.

In chapter three, I expand upon the temperature-jump detection method described in chapter two. By adapting this method to accommodate X-ray diffraction images, I demonstrate that we can detect temperature-jumps within a crystalline context. This is a crucial step in the development of a generalized perturbation for time-resolved crystallography. Given the timescale of the measurements, reading out the temperature directly from the X-ray data is the only effective way to track the sample’s response. Thus, our method offers proof-of-principle that IR laser-based temperature-jumps are feasible for time-resolved crystallography. While measuring the diffuse scattering signal is useful for temperature-jump detection, the diffuse signal also holds the potential to inform our understanding of protein dynamics.

In chapter four, I review the field of macromolecular diffuse scattering, as of late 2017. I begin by considering data collection practices, which requires extremely careful and controlled measurements. Then I examine different group's approaches to processing the data, as well as their models of the disorder that drives it. Finally, I consider the broader impact of diffuse analysis upon the field, ranging from the improvement of molecular dynamics forcefields to improved phasing and resolution extension. While these impacts hold exciting implications, it is clear that collecting high-quality is the first challenge to solve.

In chapter five, I examine the challenges of collecting high-quality diffuse scattering from protein crystals. I describe how parasitic scattering can confound our ability to develop rigorous models of the crystalline disorder that gives rise to the diffuse signal. Then I work through experimental measures that we took to minimize parasitic scattering while maximizing diffuse scatter driven by protein motions.