title: Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy.
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.
authors.affiliation: Department of Chemistry, Indiana University , 800 East Kirkwood, Bloomington, Indiana 47405, United States.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Basom
authors.affiliation: Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Korea University , Seoul 02841, Republic of Korea.|Department of Chemistry, Korea University , Seoul 02841, Republic of Korea.
authors.forename: Michał
authors.initials: M
authors.lastname: Maj
authors.affiliation: Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Korea University , Seoul 02841, Republic of Korea.|Department of Chemistry, Korea University , Seoul 02841, Republic of Korea.
authors.forename: Minhaeng
authors.initials: M
authors.lastname: Cho
authors.affiliation: Department of Chemistry, Indiana University , 800 East Kirkwood, Bloomington, Indiana 47405, United States.
authors.forename: Megan C
authors.initials: MC
authors.lastname: Thielges
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002164:Camphor / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D010649:Phenylalanine / Q000031:analogs & derivatives
chemical_list: D000409:Alanine; D002164:Camphor; D019475:Camphor 5-Monooxygenase; D009570:Nitriles; C530634:p-cyanophenylalanine; D010649:Phenylalanine