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.