Proteins undergo constant, thermally induced fluctuations in which hydrogen bonded structures unfold in events ranging from global unfolding to small scale events like the fraying of a helix. These conformational fluctuations are critical pieces of a proteins energy landscape and have been linked to protein function, but experimentally separating changes in the local energy surface, like the free energies that result in helix fraying, from the global folding stability has proved challenging, especially in membrane proteins. Membrane proteins fold and function in a unique cellular environment that makes them difficult to study yet critical to understanding biological processes and human disease. My thesis work as presented here has focused on examining and understanding protein energy landscapes with more nuance and greater detail than has previously been possible.

In Chapter 2, we describe the native conformational fluctuations of E. Coli helical membrane protein GlpG in two commonly used cell membrane mimetics as measured by HDX-MS. We show that the conformational fluctuations of helical membrane proteins are more diverse than have been previously described, and we also show that, while the cooperativity landscape appears to be maintained in both bilayers, the overall unfolding behavior is influenced by bilayer properties. This work represents a step forward in examining the cooperativity landscapes and denatured state ensembles (DSE) of helical membrane proteins and provides direct evidence that the membrane environment impacts the folding behavior and dynamics of some helical membrane proteins.

In Chapter 3, we define a new method, the Cooperativity Factor (CoF), to quantify the cooperativity of all peptides obtained in a given HDX-MS experiment. This allows us to examine the cooperativity landscape of entire proteins and draw conclusions about the relationship between protein energy landscapes and function in both membrane and soluble proteins. Specifically, we show that highly cooperative regions likely allow proteins to generate large scale motions, such as those needed to couple voltage sensing to pore opening in the ion channel KvAP. In contrast, non-cooperativity allows adjacent residues to function independently and thus create small-scale, finessed movements that make functions like ligand binding possible. We also show that single mutations can vastly alter the cooperativity landscape and that the cooperativity pattern is related to but still independent from global protein structure.

Overall, our studies deepen our understanding about how the local energy landscape relates to protein function and how the membrane environment can affect the local energy landscape of membrane proteins. Our development of new techniques also facilitates further investigation into the relationship between protein structure, cooperativity and function.