Resolving Correlated Motions in Proteins by X-ray Diffraction

Abstract

Proteins have many rotatable bonds with low energetic barriers and exist at a nanometer length scale under constant influence by the thermal fluctuations of water. Accordingly, proteins are dynamic molecules, and their motions are often essential to their proper folding, biological function, and regulation. While structural methods can characterize averaged features of macromolecules, characterizing the motions of proteins at the atomic scale with high temporal resolution remains a major challenge to developing and validating mechanistic models that describe their behavior. This dissertation presents new tools and methods to resolve correlated motions in proteins with X-ray diffraction---by understanding what parts of a protein move together, it will be possible to develop models that describe their dynamics and regulation. This is accomplished by addressing three successive aims. First, I present a software library that facilitates exploratory analysis of crystallographic data. Second, I describe and validate a data collection strategy for accurate X-ray diffraction experiments at room temperature. Finally, I use multi-temperature and electric-field-dependent X-ray diffraction experiments to characterize molecular motions in dihydrofolate reductase, and validate a mechanistic model of allosteric regulation in the enzyme's Michaelis complex. Together, these aims develop new ways to study protein dynamics with X-ray diffraction and present a case study that uses correlated motions in an enzyme to infer a mechanism of allosteric regulation.