In vitro folding of phage P22 coat protein with amino acid substitutions that confer in vivo temperature sensitivity.
The coat protein that forms the icosahedral shell of phage P22 can be efficiently refolded in vitro [Teschke, C. M., & King, J. (1993) Biochemistry 32, 10839-10847]. Temperature-sensitive mutants of coat protein interfere with folding or assembly in vivo [Gordon, C. L., & King, J. (1993) J. Biol. Chem. 268, 9358-9368]. The folding of a set of phage P22 coat proteins carrying the temperature-sensitive for folding (tsf) substitutions W48Q, A108V, G232D, T294I, and F353L has been investigated in vitro. Denatured tsf polypeptides were able to fold into soluble species with high efficiency. The efficiency of folding of the wild-type (WT) and mutant polypeptides at different temperatures showed sharp transitions where aggregation predominated over folding. The refolding of the tsf mutant proteins did not show an obvious thermal defect in yield. The tsf polypeptides folded through the long-lived kinetic intermediate previously described for WT coat protein with similar relaxation times. The folding kinetics of the tsf polypeptides in bisANS, a hydrophobic fluorescent dye, were also similar to those of the WT protein. However, the folded tsf proteins showed decreased secondary structure compared to WT coat protein. Analysis of the folded state by native gel electrophoresis revealed that the tsf coat proteins folded into dimers and trimers, not monomers. The dimer and trimer species were incompetent for assembly. Once formed, dimers and trimers showed no propensity toward aggregation. The folding pathway of the mutant polypeptides must be quite similar to the WT pathway, but at some step inappropriate intersubunit interactions occur due to the amino acid substitutions, trapping the subunits from assembly.
Teschke
C M
CM
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
King
J
J
D000817:Anilino Naphthalenesulfonates; D017100:Bacteriophage P22 / Q000737:chemistry*; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013388:Succinimides; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D013388:Succinimides; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)