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Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13
- Source :
- Biochimica et Biophysica Acta. Biomembranes 1509 (2000), Biochimica et Biophysica Acta. Biomembranes, 1509, 311-323
- Publisher :
- Elsevier Science B.V.
-
Abstract
- During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17–24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.
- Subjects :
- Genes, Viral
Protein Conformation
Lysine
Lipid Bilayers
Molecular Sequence Data
Phospholipid
Biophysics
Gene Expression
Biology
medicine.disease_cause
Biochemistry
N-terminal protein arm rearrangement
Cyclic N-Oxides
Residue (chemistry)
chemistry.chemical_compound
Capsid
Naphthalenesulfonates
medicine
Escherichia coli
M13 major coat protein
Amino Acid Sequence
Amino Acids
Cloning, Molecular
Fluorescence spectroscopy
Phospholipids
Fluorescent Dyes
Acrylamide
Lipid–protein interaction
Cell Biology
biology.organism_classification
Cysteine scanning
Membrane
Biofysica
Filamentous bacteriophage
chemistry
Cytoplasm
Mutation
biology.protein
Mutagenesis, Site-Directed
Protein G
EPS
Lipid-protein interaction
Phage assembly
Bacteriophage M13
Plasmids
Subjects
Details
- Language :
- English
- ISSN :
- 00052736
- Issue :
- 1-2
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta (BBA) - Biomembranes
- Accession number :
- edsair.doi.dedup.....3ea441e6035aab27f5dac508d7d41423
- Full Text :
- https://doi.org/10.1016/S0005-2736(00)00314-X