Conformations and Dynamics of Modular Redox Enzymes via Site-Specific 2D Infrared Spectroscopy

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Proteins need to move in order to function, but scientists still know very little about such motions. This research will allow Dr. Feng to develop a modern research tool in his own laboratory to identify how proteins move when performing specific functions. Such an empowering technique is currently lacking at the home organization. Since the University of New Mexico is a Hispanic Serving Institution, this award will provide frontline research opportunities that are rarely made available to their underrepresented minority students. The project will also provide unique research opportunities to outstanding students from other institutions, to ensure a diverse scientific workforce that remains engaged in active, cutting-edge research.Deciphering protein dynamics is a pressing challenge at the forefront of efforts to understand the underlying mechanisms of electron transfer, a fundamental process in biology. This project focuses on conformational dynamics of nitric oxide synthases (NOS), large, modular enzymes. NOSs are important because of the pivotal roles of nitric oxide production in diverse signaling processes. One hallmark of NOS is its multi-domain architecture with highly flexible tethers, allowing for dynamic, regulated interdomain electron transfer. The NOS enzyme has been extensively investigated, but due to an intrinsic complexity, involving not only dynamics, but also partner protein binding and posttranslational modification, a comprehensive picture of how it is regulated remains lacking. Quantitative insights into both the large-scale domain movements and the more localized dynamics in the docked state are necessary to provide mechanistic details. This Transitions project will enable the PI to forge a new direction in studying the docked state conformations and dynamics via site-specific 2D infrared (IR) spectroscopy. When combined with the spatial precision provided by site-selective labeling with IR probes, the approach will enable unprecedented study of the conformations and dynamics in the NOS docked states with high spatial and temporal resolution. The objective of this multidisciplinary project is to achieve residue-specific characterizations of structural dynamics of the NOS docked states and delineate their contributions to function. As NOS is a paradigm signaling system, the results will have a broad-reaching impact. The proposed 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 between domains. This project thus represents an exciting next step in studying structure-dynamics-function relationships in multidomain proteins.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.