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.