The adsorption protein of filamentous phage fd: assignment of its disulfide bridges and identification of the domain incorporated in the coat.
The mature adsorption protein (g3p) of filamentous phage fd consists of 406 amino acids. It contains eight cysteine residues in total. To determine the disulfide bond pattern, purified g3p was proteolytically digested, and the resulting peptides were separated using RP-HPLC. N-terminal sequencing and mass spectrometry of cysteine-containing fragments showed that each cysteine is involved in an intramolecular disulfide bond. The cystine sites are Cys7-Cys36, Cys46-Cys53, Cys188-Cys201, and Cys354-Cys371. In the native conformation of g3p, none of the disulfide bridges is accessible to alkylating agents after treatment with DTT. The cystine sites seem therefore to be located in the interior of the folded molecule or are shielded by components of the phage envelope. The part of g3p which is incorporated in the phage coat and hence is inaccessible to proteolytic cleavage was identified after treatment of phage particles with subtilisin: Immunoblots performed with antibody directed against g3p revealed essentially one g3p fragment with an apparent molecular weight of approximately 17,000 which resisted the proteolytic attack. The amino terminus of this peptide was determined to be glycine 254. This amino acid position correlates with the carboxy-terminal end of the second glycine-rich region (amino acids 218-256) in the primary structure of g3p. The notion that the extended carboxy-terminal g3p fragment is indeed entirely buried within the phage envelope is further supported by the fact that only polyclonal antibodies raised against purified g3p are able to react with this peptide, whereas those against phage coat-associated g3p are not.
Kremser
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Fakultät für Biologie, Universität Konstanz, Germany.
Rasched
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D000477:Alkylating Agents; D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins; D003545:Cysteine / Q000032:analysis; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004220:Disulfides / Q000032:analysis*; D017912:Inovirus; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013820:Thermolysin; D014760:Viral Fusion Proteins; D014764:Viral Proteins / Q000737:chemistry*
D000477:Alkylating Agents; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D004220:Disulfides; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D013820:Thermolysin; D003545:Cysteine