Substrate mutations that bypass a specific Cpn10 chaperonin requirement for protein folding

The bacteriophage T4 GroES homologue, gp31, in conjunction with the Escheri chia coil chaperonin GroEL, is both necessary and sufficient to fold the T4 major capsid protein, gp23, to a state competent for capsid assembly as sh own by in vivo expression studies. GroES is unable to function in this role as a productive co-chaperonin. The sequencing and characterization of muta tions within gp23 that confer GroEL and gp31 chaperonin-independent folding of the mutant protein suggest that the chaperonin requirements are due to specific sequence determinants or structures in critical regions of gp23 th at behave in an additive fashion to confer a chaperonin bypass phenotype. C onservative amino acid substitutions in these critical regions enable gp23 to fold in a GroEL-gp31 chaperonin-independent mode, albeit less efficientl y than wild type, both in vivo and in vitro. Although the presence of funct ional GroEL-gp31 enhances folding of the mutated gp23 in vivo, GroEL-GroES has no such effect. Site-directed mutagenesis experiments suggest that a tr anslational pausing mechanism is not responsible for the bypass mutant phen otype. Polyhead reassembly experiments are also consistent with direct, pos t-translational effects of the bypass mutations on polypeptide folding. Giv en our finding that gp31 is not required for the binding of the major capsi d protein to GroEL and that active GroES is incapable of folding the gp23 p olypeptide chain to native conformation, our results suggest co-chaperonin specificity in the folding of certain substrates.