Abstract
The bacteriophage T4 GroES homologue, gp31, in conjunction with the Escherichia coli chaperonin GroEL, is both necessary and sufficient to fold the T4 major capsid protein, gp23, to a state competent for capsid assembly as shown by in vivo expression studies. GroES is unable to function in this role as a productive co-chaperonin. The sequencing and characterization of mutations 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 that behave in an additive fashion to confer a chaperonin bypass phenotype. Conservative amino acid substitutions in these critical regions enable gp23 to fold in a GroEL-gp31 chaperonin-independent mode, albeit less efficiently than wild type, both in vivo and in vitro. Although the presence of functional GroEL-gp31 enhances folding of the mutated gp23 in vivo, GroEL-GroES has no such effect. Site-directed mutagenesis experiments suggest that a translational pausing mechanism is not responsible for the bypass mutant phenotype. Polyhead reassembly experiments are also consistent with direct, post-translational effects of the bypass mutations on polypeptide folding. Given our finding that gp31 is not required for the binding of the major capsid protein to GroEL and that active GroES is incapable of folding the gp23 polypeptide chain to native conformation, our results suggest co-chaperonin specificity in the folding of certain substrates.
Highlights
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201-1503
Before 1970, it was determined that bacteriophage T4 synthesizes a bacteriophage protein that is required to assemble its major capsid protein, gp23, into head structures; without this factor what would be called inclusion bodies of the major capsid protein accumulate in vivo precluding the formation of proheads [23]
Identification of Essential Components Required for the Proper Folding and Oligomerization of the T4 Major Capsid Protein—In order to study the role of gp31 as a T4 co-chaperonin, T4 genes 23 and 31 were cloned independently into the pET3a expression vector
Summary
Materials—Molecular biology reagents were purchased from either Boehringer Mannheim, New England Biolabs, or Life Technologies, Inc. PTrc99A (Amersham Pharmacia Biotech) and pET3a-derived plasmids (Novagen) were transformed and expressed in HMS174(DE3), BL21(DE3), and their non-DE3 derivatives (Novagen). Plasmid Construction and Site-directed Mutagenesis—Constructs were made by inserting the target T4 gene downstream of the T7 promoter in either the pET3a (Novagen) expression vector using the NdeI and BamHI restriction sites or the pTrc99A (Amersham Pharmacia Biotech) expression vector using the XbaI restriction endonuclease site. Constructs were made by inserting the target T4 gene into the pET3a (Novagen) expression vector, creating pET23 and pETBY, respectively. Isolating an AseIBglII fragment containing gene 31 from M13mp11-TR5 and directly cloning into the pET3a vector as described above [30] constructed pET31. An XbaI fragment from this vector, containing gene 31 and its Shine-Delgarno sequence, was directly cloned into pTrc99A (Amersham Pharmacia Biotech), creating pTrc. Rabbit antibodies directed against gp were used in conjunction with the chemiluminescent ECL technique (Amersham Pharmacia Biotech) for detection of gp protein
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