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Abstract
Proteins are not static structures; they are dynamic machines whose motions underpin nearly all aspects of biological function. While structure prediction tools such as AlphaFold have revolutionized biology and made atomic level protein structures widely accessible, understanding the landscapes of folding, flexibility, and mechanical behavior remains a major area of investigation. This dissertation presents a set of experimental and computational work illuminating protein conformational dynamics as a way of understanding protein function. First, we investigated the unfolding dynamics of the final four extracellular domains of cadherin-23 (CDH23EC25–MAD28), a protein essential for hearing. Using all-atom simulations, we saw that the membrane-adjacent domain (MAD28) of cadherin-23 unfolds first under tension, but using hydrogen-deuterium exchange mass spectrometry we concluded that the unfolding behavior is likely driven by the topology of the folded MAD28 rather than a difference of thermodynamic stability. We go on to use calcium depletion and two deafness-causing mutations to show that instability in ECs will cascade into MAD28, but that destabilization of MAD28 alone does not affect ECs, likely due to stabilizing interactions in EC27. These insights represent the first experimental exploration of MAD (in)stability and present a molecular basis for the impact domain unfolding has on healthy and diseased CDH23 function. In parallel, we used simulations to study insulin-degrading enzyme, discovering that it engages with substrates using “beta grab” interactions, that it undergoes a grinding motion to promote substrate unfolding, and that targeted mutations can tune these functions. We also introduce DynaLab, a computational notebook to run and analyze coarse-grained simulations in Upside that was built to make protein dynamics more accessible. Collectively, the insights presented here offer a deeper understanding of how critical motions are for protein function and how we can deploy available tools to demystify protein conformational dynamics.