Two dimensional infrared spectroscopic investigations of protein and enzyme dynamics

Abstract

Ultra fast two dimensional infrared (2D IR) spectroscopy is used to measure dynamics and interactions in complex materials in the ground electronic state under thermal equilibrium conditions. Two observables, ultra fast chemical exchange and spectral diffusion, will be discussed. In a 2D IR experiment, three variably time delayed IR excitation pulses, tuned to the vibrational transition of interest, give rise to a fourth vibrational echo pulse, the signal in the experiment. The first two pulses in the sequence label the vibrations with their initial frequencies. After a waiting period, Tw, the third pulse induces the vibrational echo, which reads out the final vibrational frequencies. Structural changes of the system cause the vibrational frequencies to change. Therefore, measuring the time evolution of the vibrational frequencies reports on the time evolution of the structure. Folded proteins can exist in multiple conformational substates. Each substate reflects a local minimum on the free‐energy landscape with a distinct structure. Using 2D‐IR chemical exchange spectroscopy, conformational switching between two well defined substates of two myoglobin mutants were observed on the ~50 ps time scale. The conformational dynamics were measured through the growth of off‐diagonal peaks in the 2D IR spectra of CO bound to the heme. The conformational switching likely involves motion of the distal histidine/E helix that changes the position of the histidine's imidazole side group. These results demonstrate that interconversion between protein conformational substates can occur on very fast time scales. Detailed MD simulations are able to capture some of the observed behavior, but not quantitatively. Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. 2D IR spectroscopy was used to measure the dynamics of cyt P450cam from Pseudomonas putida using CO bound at the active site as the vibrational probe. The experiments measure spectral diffusion, the time evolution of the CO vibrational frequencies within the in homogeneously broadened vibrational absorption line. The time evolution of the frequencies is caused by the time evolution of the enzyme structure. The enzyme bound to both its natural substrate, camphor, and a series of related substrates were investigated to explicate the role of dynamics in molecular recognition in cyt P450cam and to delineate how the motions may contribute to hydroxylation specificity. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of picosecond time scale contribute to the variation in activity of cyt P450cam toward different substrates.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.