Functional Protein Conformations and Dynamics via Transparent Window 1D & 2D Infrared Spectroscopy

Project Summary/Abstract Proteins are the molecular machines of a cell that orchestrate virtually all processes. Like a macroscopic machine, their function requires that they move to adopt different states. Thus, a modern view of protein biophysics has expanded from the structure-function paradigm to include dynamics - population of ensembles of protein states (conformational heterogeneity) and their interconversion. Fully delineating the dynamics that underlie function is however complicated by the immense complexity of proteins, due to both their large size with spatial heterogeneity of their chemistry and the broad timescales over which states may interconvert, ranging from large-scale processes such as aggregation that occur over days to years to the picosecond fluctuations of side chains and solvent. Among these scales, the local small-scale changes in proteins that involve rapidly interconverting states are perhaps among the most challenging to characterize and least well understood. However, such motions are argued to be central to many aspects of protein function, such as main contributors to the entropy of reactions, allosteric communication within domains, catalytic and binding specificity, et al. This research program is directed at developing rigorous methodologies to advance understanding of fundamental protein biophysics in important biological processes. We are developing infrared (IR) spectroscopy as an approach with inherently high spatial and temporal resolution for accessing all involved conformational ensembles and dynamics. By incorporating vibrational groups with frequencies within a transparent window of protein IR spectra we avoid the complexity of spectral congestion to enable inspection of single vibrational modes at local sites anywhere throughout proteins. We are combining modern approaches in biochemistry for selective labeling with state-of-the-art methods in multidimensional spectroscopy to provide rigorous analysis of frequency heterogeneity, coupling, and dynamics. The application and development of new biochemical and spectroscopic methods should elucidate the biophysical foundations of protein function with unprecedented detail, providing information with promise to buttress advances in broad areas of biology and medicine. In addition, through execution of this project, undergraduate, graduate, and postdoctoral researchers will be broadly trained in multidisciplinary science, strengthening the nation’s scientific workforce.