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Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy.

ABSTRACT: Enzymes move on a variety of length and time scales. While much is known about large structural fluctuations that impact binding of the substrates and release of products, little is known about faster motions of enzymes and how these motions may influence enzyme-catalyzed reactions. This Letter reports frequency fluctuations of the azide anion bound to the active site of formate dehydrogenase measured via 2D IR spectroscopy. These measurements reveal an underdamped oscillatory component to the frequency-frequency correlation function when the azide is bound to the NAD(+) ternary complex. This oscillation disappears when the reduced cofactor is added, indicating that the oscillating contributions most likely come from the charged nicotinamide ring. These oscillatory motions may be relevant to donor-acceptor distance sampling of the catalyzed hydride transfer and therefore may give future insights into the dynamic behavior involved in enzyme catalysis.

SUBMITTER: Pagano P

PROVIDER: S-EPMC4939886 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy.

Pagano Philip P Guo Qi Q Kohen Amnon A Cheatum Christopher M CM

The journal of physical chemistry letters 20160621 13

Enzymes move on a variety of length and time scales. While much is known about large structural fluctuations that impact binding of the substrates and release of products, little is known about faster motions of enzymes and how these motions may influence enzyme-catalyzed reactions. This Letter reports frequency fluctuations of the azide anion bound to the active site of formate dehydrogenase measured via 2D IR spectroscopy. These measurements reveal an underdamped oscillatory component to the f ...[more]

PMID: 27305279

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Project description:Proteins, enzymes, and other biological molecules undergo structural dynamics as an intrinsic part of their biological functions. While many biological processes occur on the millisecond, second, and even longer time scales, the fundamental structural dynamics that eventually give rise to such processes occur on much faster time scales. Many decades ago, chemical kineticists focused on the inverse of the reaction rate constant as the important time scale for a chemical reaction. However, through transition state theory and a vast amount of experimental evidence, we now know that the key events in a chemical reaction can involve structural fluctuations that take a system of reactants to its transition state, the crossing of a barrier, and the eventual relaxation to product states. Such dynamics occur on very fast time scales. Today researchers would like to investigate the fast structural fluctuations of biological molecules to gain an understanding of how biological processes proceed from simple structural changes in biomolecules to the final, complex biological function. The study of the fast structural dynamics of biological molecules requires experiments that operate on the appropriate time scales, and in this Account, we discuss the application of ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy to the study of protein dynamics. The 2D IR vibrational echo experiment is akin to 2D NMR, but it operates on time scales many orders of magnitude faster. In the experiments, a particular vibrational oscillator serves as a vibrational dynamics probe. As the structure of the protein evolves in time, the structural changes are manifested as time-dependent changes in the frequency of the vibrational dynamics probe. The 2D IR vibrational echo experiments can track the vibrational frequency evolution, which we then relate to the time evolution of the protein structure. In particular, we measured protein substate interconversion for mutants of myoglobin using 2D IR chemical exchange spectroscopy and observed well-defined substate interconversion on a sub-100 ps time scale. In another study, we investigated the influence of binding five different substrates to the enzyme cytochrome P450(cam). The various substrates affect the enzyme dynamics differently, and the observed dynamics are correlated with the enzyme's selectivity of hydroxylation of the substrates and with the substrate binding affinity.

Project description:We have studied the structure of (Ala)5, a model unfolded peptide, using a combination of 2D IR spectroscopy and molecular dynamics (MD) simulation. Two different isotopomers, each bis-labeled with (13)C?O and (13)C?(18)O, were strategically designed to shift individual site frequencies and uncouple neighboring amide-I' modes. 2D IR spectra taken under the double-crossed ??/4, -?/4, Y, Z? polarization show that the labeled four-oscillator systems can be approximated by three two-oscillator systems. By utilizing the different polarization dependence of diagonal and cross peaks, we extracted the coupling constants and angles between three pairs of amide-I' transition dipoles through spectral fitting. These parameters were related to the peptide backbone dihedral angles through DFT calculated maps. The derived dihedral angles are all located in the polyproline-II (ppII) region of the Ramachandran plot. These results were compared to the conformations sampled by Hamiltonian replica-exchange MD simulations with three different CHARMM force fields. The C36 force field predicted that ppII is the dominant conformation, consistent with the experimental findings, whereas C22/CMAP predicted similar population for ?+, ?, and ppII, and the polarizable Drude-2013 predicted dominating ? structure. Spectral simulation based on MD representative conformations and structure ensembles demonstrated the need to include multiple 2D spectral features, especially the cross-peak intensity ratio and shape, in structure determination. Using 2D reference spectra defined by the C36 structure ensemble, the best spectral simulation is achieved with nearly 100% ppII population, although the agreement with the experimental cross-peak intensity ratio is still insufficient. The dependence of population determination on the choice of reference structures/spectra and the current limitations on theoretical modeling relating peptide structures to spectral parameters are discussed. Compared with the previous results on alanine based oligopeptides, the dihedral angles of our fitted structure, and the most populated ppII structure from the C36 simulation are in good agreement with those suggesting a major ppII population. Our results provide further support for the importance of ppII conformation in the ensemble of unfolded peptides.

Dataset Information

Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy.

Publications

Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy.

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