Dynamics at Specific Sites in Proteins Studied by 2D IR Vibrational Echo Spectroscopy

Abstract

The techniques of two-dimensional infrared (2D IR) spectroscopy enable the study of fluctuations of molecules and their environments on fast timescales. Characterization of specific sites in proteins requires the use of extrinsic vibration probes to alleviate the spectral complexity that hinders the application of IR spectroscopy with protein samples. Heme-bound CO has been a popular vibrational probe of protein active sites and, for example, was employed to characterize the involvement of dynamics in the specificity of cytochrome P450 using 2D IR spectroscopy. In efforts toward extending the 2D IR studies beyond heme proteins, an azidophenylalanine vibrational probe was introduced at a specific site within myoglobin using orthogonal transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs evolved to insert azidophenylalanine in response to an amber codon. The azidophenylalanine vibrational probe was placed within the heme pocket at Phe43, permitting investigation of one site in myoglobin via two different vibrational probes - the genetically introduced azidophenylalanine and heme bound CO. The use of two probes allowed assessment of the perturbation due to introduction of each and the measurement of the heme pocket dynamics via two perspectives, showing that the reported dynamics were reflective of the protein motions, and not intrinsic to a particular probe. This approach can be used in the future to characterize the dynamics at virtually any site throughout virtually any protein.