The Adsorption Protein of Filamentous Phage fd: Assignment of Its Disulfide Bridges and Identification of the Domain Incorporated in the Coat

Abstract

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.

Colicin Biology

“…As discussed in Colicin Reception, binding and translocation require the presence of the minor coat capsid protein g3p (or pIII), which is located at the tip of the bacteriophage particle (182,234). Interestingly, like colicins, this protein is organized into three distinct domains separated by flexible glycine-rich linkers, each of them involved in a specific stage of the infection process (361,443,612) (see structural organization section). The functional features of the similar organizations between colicins and g3p proteins were discovered by the construction of active chimeras between the M13 g3p protein and colicin E3 (301).…”

The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3

“…The N-terminal part can be divided into two different domains, N1 and N2, mediating penetration and adsorption during infection, respectively (1,30). The C-terminal part is responsible for the interaction with g6p, thereby anchoring the g3p in the membrane of the phage coat (1,14,21). Furthermore, the g3p has a crucial role in the assembly of the filamentous phage, since in the absence of g3p, the proper termination of the assembly and the following release of the phage will not take place (24).…”

A method for directed evolution and functional cloning of enzymes

“…To apply this method to proteins expressed on filamentous phage, the three single cysteines of the pVI, pVII, and pIX coat proteins first were mutagenized to alanine. The eight buried cysteine residues in the pIII protein were left unchanged, as they likely form structurally important disulfide bridges (18,19). Unfortunately, repeated attempts to selectively modify unique cysteine residues introduced near the active site of several enzymes displayed on phage, by either disulfide exchange, maleimide addition, or alkylation reactions, resulted in significant nonspecific labeling of phage coat proteins.…”