Structural characterization of membrane insertion of M13 procoat, M13 coat, and Pf3 coat proteins.
A new, simple, and efficient purification method has been developed for the extremely hydrophobic M13 procoat, M13 coat, and Pf3 coat proteins. Homogeneous preparations were obtained in 2-propanol/0.1% TFA, where M13 coat protein is found to be dissolved in a monomeric form, and the two other proteins as dimers or trimers. The conformations of these particular proteins in different environments have been determined by circular dichroism and infrared spectroscopy. In organic solvents, the proteins adopt a conformation with an average helix content of 90%. In lipid bilayers composed of phosphatidylcholine and phosphatidylglycerol lipids, the average helix content is 50% for M13 procoat protein, 60% for M13 coat protein, and 75% for Pf3 coat protein. The orientational order parameter S alpha of the protein helices in planar lipid bilayers have been determined by polarized infrared measurements in the amide I spectral range. The helices of the three proteins are oriented preferentially parallel to the membrane normal, with S alpha = 0.63 for M13 procoat protein, S alpha = 0.58 for Pf3 coat protein, and a distinctly higher value of S alpha = 0.81 for M13 coat protein.
Thiaudière
E
E
Institute of Physical Chemistry, Swiss Federal Institute of Technology, Lausanne.
Soekarjo
M
M
Kuchinka
E
E
Kuhn
A
A
Vogel
H
H
D000595:Amino Acid Sequence; D017104:Bacteriophage M13; D002213:Capsid / Q000737:chemistry* / Q000302:isolation & purification; D036022:Capsid Proteins; D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000302:isolation & purification; D017105:Pseudomonas Phages; D011550:Pseudomonas aeruginosa; D017550:Spectroscopy, Fourier Transform Infrared
D036022:Capsid Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011498:Protein Precursors; C025668:coat protein, Bacteriophage M13; C084332:major coat protein, Pseudomonas phage Pf3