Folding of the phage P22 coat protein in vitro.
Within infected Salmonella cells, newly synthesized 47-kDa phage P22 coat polypeptides fold without covalent modifications into assembly-competent subunits. Coat protein subunits interact with scaffolding protein to form the icosahedral procapsid precursor of the mature, T = 7, virions. In these lattices, the coat subunits form seven classes of local bonding interactions [Prasad, B. V. V., Prevelige, P. E., Marieta, E., Chen, R. O., Thomas, D., King, J., & Chiu, W. (1993) J. Mol. Biol. 231, 65-74]. Coat protein denatured in guanidine hydrochloride could be refolded to soluble, monomeric subunits by rapid dilution into buffer at concentrations of protein up to 25 micrograms/mL. The fluorescence emission spectrum of soluble coat protein monomers was between that of the assembled shells and the denatured protein, suggesting the presence of tryptophans at the subunit interfaces in the shells. Kinetic studies of the refolding of coat protein revealed an intermediate whose continued folding could be inhibited by the hydrophobic dye bisANS. The kinetic intermediate bound 10.80 +/- 1.20 bisANS molecules while the folded monomer bound 1.24 +/- 0.36 bisANS molecules. When coat polypeptide chains were refolded at 50 micrograms/mL, aggregation competed with folding. Aggregation of the folding intermediates increased in the presence of bisANS. The kinetic folding intermediate that binds bisANS probably represents the species at the junction of the productive pathway to soluble and assembly-competent coat monomers and the off-pathway steps to inclusion bodies. The relationship between these soluble monomers and the conformations observed in the T = 7 lattice remains unclear.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
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Teschke
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King
D000817:Anilino Naphthalenesulfonates; D017100:Bacteriophage P22 / Q000378:metabolism*; D002213:Capsid / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D012486:Salmonella typhimurium / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006146:Guanidines; D046911:Macromolecular Substances; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D019791:Guanidine