Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy

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Title
Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy
Author(s)
Edward J. Basom; Michał Maj; Minhaeng Cho; Megan C. Thielges
Publication Date
2016-06
Journal
ANALYTICAL CHEMISTRY, v.88, no.12, pp.6598 - 6606
Publisher
AMER CHEMICAL SOC
Abstract
Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast time scale and small chromophores of infrared (IR) spectroscopy are well-suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated p-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. Molecular dynamics (MD) simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function. © 2016 American Chemical Society
URI
https://pr.ibs.re.kr/handle/8788114/2750
ISSN
0003-2700
Appears in Collections:
Center for Molecular Spectroscopy and Dynamics(분자 분광학 및 동력학 연구단) > Journal Papers (저널논문)
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