title: Folding and stability of mutant scaffolding proteins defective in P22 capsid assembly.
abstract: Bacteriophage P22 scaffolding subunits are elongated molecules that interact through their C termini with coat subunits to direct icosahedral capsid assembly. The soluble state of the subunit exhibits a partially folded intermediate during equilibrium unfolding experiments, whose C-terminal domain is unfolded (Greene, B., and King, J. (1999) J. Biol. Chem. 274, 16135-16140). Four mutant scaffolding proteins exhibiting temperature-sensitive defects in different stages of particle assembly were purified. The purified mutant proteins adopted a similar conformation to wild type, but all were destabilized with respect to wild type. Analysis of the thermal melting transitions showed that the mutants S242F and Y214W further destabilized the C-terminal domain, whereas substitutions near the N terminus either destabilized a different domain or affected interactions between domains. Two mutant proteins carried an additional cysteine residue, which formed disulfide cross-links but did not affect the denaturation transition. These mutants differed both from temperature-sensitive folding mutants found in other P22 structural proteins and from the thermolabile temperature-sensitive mutants described for T4 lysozyme. The results suggest that the defects in these mutants are due to destabilization of domains affecting the weak subunit-subunit interactions important in the assembly and function of the virus precursor shell.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: B
authors.initials: B
authors.lastname: Greene
authors.forename: J
authors.initials: J
authors.lastname: King
mesh_terms: D017100:Bacteriophage P22 / Q000502:physiology*; D002213:Capsid / Q000378:metabolism*; D002942:Circular Dichroism; D009113:Muramidase / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D019065:Virus Assembly
chemical_list: D015678:Viral Structural Proteins; C111088:scaffolding protein, bacteriophage P22; D014364:Tryptophan; D009113:Muramidase