title: Membrane assembly of the bacteriophage Pf3 major coat protein.
abstract: The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.
authors.affiliation: Laboratory of Molecular Physics, Department of Biomolecular Sciences, Wageningen University and Research Center, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
authors.forename: A B
authors.initials: AB
authors.lastname: Meijer
authors.forename: R B
authors.initials: RB
authors.lastname: Spruijt
authors.forename: C J
authors.initials: CJ
authors.lastname: Wolfs
authors.forename: M A
authors.initials: MA
authors.lastname: Hemminga
mesh_terms: D000595:Amino Acid Sequence; D017104:Bacteriophage M13; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D036022:Capsid Proteins; D003545:Cysteine / Q000235:genetics; D017912:Inovirus; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D017105:Pseudomonas Phages / Q000737:chemistry* / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000821:virology; D013050:Spectrometry, Fluorescence; D019065:Virus Assembly / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D008565:Membrane Proteins; D009282:Naphthalenesulfonates; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; D010743:Phospholipids; C025668:coat protein, Bacteriophage M13; C071283:coat protein, Pseudomonas phage Pf1; C094877:dioleoyl phosphatidylethanolamine; C006422:1,8-I-AEDANS; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; C084332:major coat protein, Pseudomonas phage Pf3; C017251:1,2-oleoylphosphatidylcholine; D003545:Cysteine