Protein conformational heterogeneity, motion, flexibility are thought to significantly impact protein function, but investigation of their contribution is limited by the availability of methods for characterizing rapidly fluctuating protein states with both high spatial and temporal resolution.  Multidimensional infrared spectroscopy is emerging as a powerful technique that directly probes the structural dynamics of proteins on fast timescales.  However, the spectral congestion inherent to protein spectra limits application of infrared spectroscopy to the study of protein systems.  We incorporate into proteins vibrational probes with spectrally isolated frequencies and local-mode character that make possible rigorous analysis of protein environments and dynamics with infrared spectroscopy.  In particular, both heme-bound carbon monoxide and nitriles selectively incorporated as unnatural amino acids were used as probes of cytochrome P450.  The application of these methods to investigate the contribution of protein dynamics and conformational heterogeneity to the specificity of its catalytic activity will be discussed.  In addition, studies of mutants of cytochrome P450 shed light on the specific protein residues that contribute to the protein's dynamics.

Characterization of protein dynamics and conformational heterogeneity with 2D infrared spectroscopy

246th National Meeting (2013)

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