title: A new approach to the study of protein-protein interaction by FTIR: complex formation between cytochrome P450BM-3 heme domain and FMN reductase domain.
abstract: We describe a new approach to the study of protein-protein interaction using Fourier transform infrared spectroscopy (FTIR). This approach is based on the combination of FTIR technique with both protein titration experiments and the principal component analysis (factor analysis) of the IR absorption spectra in the 1500-1800 cm(-1) region for the protein mixtures. We have applied this approach to the interaction of the heme domain with the FMN domain of bacterial monooxygenase cytochrome P450BM-3 (CYP102A1). The analysis reveals that the first principal component reflects the protein-protein complex formation because the loading factors show a clear systematic dependence on the concentration of the heme domain according to a titration curve with a dissociation constant of approximately 5 microM. The spectrum of the first principal component has been assigned to structural changes in the secondary structure (increase of beta-sheet and alpha-helix and decrease of turn structures), amino acid side chains (protonation of aspartate and C-terminal COO group), and deprotonation of a propionic acid COOD group in the heme.
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, Protein Dynamics Laboratory, 13125 Berlin, Germany.
authors.forename: Andrei
authors.initials: A
authors.lastname: Kariakin
authors.forename: Dimitri
authors.initials: D
authors.lastname: Davydov
authors.forename: Julian A
authors.initials: JA
authors.lastname: Peterson
authors.forename: Christiane
authors.initials: C
authors.lastname: Jung
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005415:Flavins; D005456:Fluorescent Dyes / Q000737:chemistry; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008861:Microsomes; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases