The folded conformation of phage P22 coat protein is affected by amino acid substitutions that lead to a cold-sensitive phenotype.
Three cold-sensitive mutants in phage P22 coat protein have been characterized to determine the effects of the amino acid substitutions that cause cold sensitivity on the folding pathway and the conformation of refolded coat protein. Here we find that the three cold-sensitive mutants which have the threonine residue at position 10 changed to isoleucine (T10I), the arginine residue at position 101 changed to cysteine (R101C), or the asparagine residue at position 414 changed to serine (N414S) were capable of folding from a denatured state into a soluble monomeric species, but in each case, the folded conformation was altered. Changes in the kinetics of folding were observed by both tryptophan and bisANS fluorescence. In contrast to the temperature-sensitive for folding coat protein mutants which can be rescued at nonpermissive temperatures in vivo by the overproduction of molecular chaperones GroEL and GroES [Gordon, C. L., Sather, S. K., Casjens, S., & King, J. (1994) J. Biol. Chem. 269, 27941-27951], the folding defects associated with the cold-sensitive amino acid substitutions were not recognized by GroEL and GroES.
Fong
D G
DG
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269, USA.
Doyle
S M
SM
Teschke
C M
CM
D000817:Anilino Naphthalenesulfonates; D017100:Bacteriophage P22 / Q000737:chemistry* / Q000235:genetics; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012486:Salmonella typhimurium / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D018835:Chaperonin 10; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D006146:Guanidines; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D019791:Guanidine