Defining Functional Dynamics of Multidomain Redox Enzymes

Project Summary Multidomain proteins make up a large portion of the human proteome. Protein dynamics due to flexible linkers connecting relatively rigid domains enable diverse biological functions, including electron transfer. Protein electron transfer lies at the heart of life. Traditional biophysical approaches have generated valuable "snapshots" of protein static states. However, multidomain proteins are best represented as a collection of accessible, dynamic architectures. Both conformational dynamics and mechanisms of regulation remain largely unclear, as the large size and conformational heterogeneity of multidomain, modular proteins pose significant challenges to most individual structural dynamics techniques, necessitating integrative investigations. The nitric oxide synthase (NOS) enzymes exemplify the complexity of modular proteins with elaborate interplay of inter-domain conformational dynamics and regulatory mechanisms. NOSs are crucial for diverse signaling processes and disease states, and understanding their regulation holds promise for therapeutic development. The study of NOS has been quite difficult because its regulation involves not only dynamics but also partner protein binding and post-translational modifications. The conformational control mechanism remains largely uncharted. We have overcome several obstacles, demonstrating a strong record of prior accomplishment in understanding large-scale conformational change. However, a comprehensive understanding will remain incomplete without quantitative insights into more localized dynamics in the docked state. Additionally, despite the abundant functional importance, the molecular mechanism of NOS regulation by phosphorylation remains largely unclear. In this new synergistic effort, we will extend our current research on large-scale dynamics to include the docked interdomain complex and explore more intricate systems in two research themes. The first theme is on functional protein dynamics, emphasizing site-specific IR spectroscopic studies of the conformations and dynamics of the docked states, as well as the mechanistic roles of phosphorylation and intrinsically disordered regions in biasing the conformational landscape. Within the second theme of method development, we will refine an approach combining quantitative cross-linking mass spectrometry and AlphaFold prediction to probe the changes in structural dynamics of homodimeric NOS proteins in response to external cues. This integrated approach draws on the unique synergistic expertise of our team. Our interdisciplinary R35 research program is thus expected to accomplish an unprecedented understanding of how regulatory interactions and modifications influence the conformational dynamics to fine-tune the protein activity, with the potential to bolster advances in broad areas of biology and medicine. The proposed research approach will also provide a blueprint for studying other complex, dynamic protein systems in which its function is gated by conformational control of electron transfer. In addition, undergraduate, graduate, and post-doc researchers will be broadly trained in multidisciplinary science, strengthening the nation’s scientific workforce.