UC Berkeley

Proteins are large, complex macromolecules that play a wide variety of essential roles in livingorganisms. It has long been appreciated that the amino acid sequence of a protein encodes its three-dimensional structure, which is essential for biological function. It is becoming increasingly appreciated that protein structure is not static; proteins are dynamic molecules, occupying many conformations with varying populations on a broad range of timescales. This conformational ensemble can be thought of as an energy landscape, and be described using the language of kinetics and thermodynamics. To truly understand how proteins execute their broad set of functions we need to understand how these energy landscapes are encoded by protein sequence, how they determine protein function, and how they are influenced by biological environments. Covalent labeling methods are ideal tools for answering these questions, as the chemical details of different covalent labeling reactions make them sensitive to protein structure, stability, and dynamics, and the temporal separation between labeling and detection facilitates the use of these methods on complex mixtures of proteins and other macromolecules. In this work, I use multiple covalent labeling methods to map the details of protein energy landscapes. First, I provide a background on protein conformational ensembles, their timescales, and on the covalent labeling methods used in this work. Second, I discuss my developments using a combination of hydroxyl radical footprinting mass spectrometry (HRF-MS) and chemical denaturation to extend our ability to measure protein global thermodynamic stability to a broader range of proteins and solution conditions. Third, I report on our use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe previously unknown conformational heterogeneity of the SARS-CoV-2 spike protein and detail how this heterogeneity is modulated by temperature, sequence, receptor binding, and interaction with antibodies. Finally, I describe work using a combination of labeling methods including HDX/MS, thiol labeling, and active site labeling to determine how the folding trajectory of the protein HaloTag is altered by translation. Lastly, I explain early efforts to extend these approaches to obtain more detailed structural information on the folding of proteins during translation.