title: Polarized ATR-FTIR spectroscopy of the membrane-embedded domains of the particulate methane monooxygenase.
abstract: The particulate methane monooxygenase (pMMO) of Methylococcus capsulatus (Bath) is an integral membrane protein that catalyzes the conversion of methane to methanol. To gain some insight into the structure-reactivity pattern of this protein, we have applied attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to investigate the secondary structure of the pMMO. The results showed that ca. 60% of the amino acid residues were structured as alpha-helices. About 80% of the peptide residues were estimated to be protected from the amide (1)H/(2)H exchange during a 21 h exposure to (2)H(2)O. In addition, a significant portion of the protein was shown to be sequestered within the bilayer membrane, protected from trypsin proteolysis. The ATR-FTIR difference spectrum between the intact and the proteolyzed pMMO-enriched membranes revealed absorption peaks only in the spectral regions characteristic for unordered and beta-structures. These observations were corroborated by amino acid sequence analysis of the pMMO subunits using the program TransMembrane topology with a Hidden Markov Model: 15 putative transmembrane alpha-helices were predicted. Finally, an attempt was also made to model the three-dimensional folding of the protein subunits from the sequence using the Protein Fold Recognition Server based on the 3D Position Specific Scoring Matrix Method. The C-terminal solvent-exposed sequence (N255-M414) of the pMMO 45 kDa subunit was shown to match the beta-sheet structure of the multidomain cupredoxins. We conclude on the basis of this ATR-FTIR study that pMMO is an alpha-helical bundle with ca. 15 transmembrane alpha-helices embedded in the bilayer membrane, together with a water-exposed domain comprised mostly of beta-sheet structures similar to the cupredoxins.
authors.affiliation: Institute of Chemistry, Academia Sinica, Nankang, Taipei, 11529 Taiwan.
authors.forename: Madhuri S
authors.initials: MS
authors.lastname: Vinchurkar
authors.forename: Kelvin H-C
authors.initials: KH
authors.lastname: Chen
authors.forename: Steve S-F
authors.initials: SS
authors.lastname: Yu
authors.forename: Shan-Jen
authors.initials: SJ
authors.lastname: Kuo
authors.forename: Hui-Chi
authors.initials: HC
authors.lastname: Chiu
authors.forename: Shu-Hua
authors.initials: SH
authors.lastname: Chien
authors.forename: Sunney I
authors.initials: SI
authors.lastname: Chan
mesh_terms: D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020619:Methylococcus capsulatus / Q000201:enzymology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry
chemical_list: D000577:Amides; D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010105:Oxygenases; C021555:methane monooxygenase; D014357:Trypsin