Role for Cysteine Residues in the In Vivo Folding and Assembly of the Phage P22 Tailspike

 

The predominantly ß-sheet P22 tailspike contains 8 reduced cysteines/666 residue per chain, which are buried and unreactive in the native trimer. Thought the native tailspike lacks disulfide bonds, transient interchain disulfide bonds are formed and reduced in the pathway to the native trimer both in vivo and in vitro (Robinson & King, 1997). These bonds occur in the protrimer, an intermediate in the formation of the interdigitated ß-sheets, that are a distinctive structural feature of the trimeric tailspike.

Each of the 8 cysteines was replaced with serine by site-specific mutagenesis in the plasmid encoded gene and expressed rom pET vectors in E. coli. Though the yields of correctly folded and assembled Cys>Ser proteins retained their thermostability. When tested for their biological activity conversion of phage capsids to infectious virus - they all exhibited wildtype activity. Thus these cysteine thiols are not required for the stability of activity of the native state.

The in vivo folding and assembly of the mutant chains was examined as a function of temperature. Five of the mutant substitutions - C169S, C267S, C287S, C613S and C635S - were impaired in their folding or assembly at higher temperature, and three were significantly impaired even at lower temperatures.

Since the trimeric states of the Cys>Ser substituted chains were as stable and active as wildtype, the impairment of tailspike formation presumably reflects problems in the in vivo folding or assembly pathways. The formation or reduction of the transient interchain disulfide bonds in the protrimer, may be the locus of these kinetic functions.

 

 

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