Polarized ATR-FTIR spectroscopy of the membrane-embedded domains of the particulate methane monooxygenase.
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.
Vinchurkar
Madhuri S
MS
Institute of Chemistry, Academia Sinica, Nankang, Taipei, 11529 Taiwan.
Chen
Kelvin H-C
KH
Yu
Steve S-F
SS
Kuo
Shan-Jen
SJ
Chiu
Hui-Chi
HC
Chien
Shu-Hua
SH
Chan
Sunney I
SI
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
D000577:Amides; D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010105:Oxygenases; C021555:methane monooxygenase; D014357:Trypsin