title: Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain.
abstract: The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the β-barrel fold. A hydrogen-bonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitive-folding mutation. The pKa of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ∼4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1-2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo By contrast, the mutants have only minor effects on capsid expansion or stability in vitro The effects of the Asp-302-His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits.
authors.affiliation: From the Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269.
authors.forename: Christina
authors.initials: C
authors.lastname: Harprecht
authors.affiliation: From the Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269.
authors.forename: Oghenefejiro
authors.initials: O
authors.lastname: Okifo
authors.affiliation: From the Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Robbins
authors.affiliation: From the Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269.
authors.forename: Tina
authors.initials: T
authors.lastname: Motwani
authors.affiliation: From the Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269 andrei.alexandrescu@uconn.edu.
authors.forename: Andrei T
authors.initials: AT
authors.lastname: Alexandrescu
authors.affiliation: From the Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269 carolyn.teschke@uconn.edu.
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Teschke
mesh_terms: D019943:Amino Acid Substitution; D017100:Bacteriophage P22 / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D036022:Capsid Proteins; D011994:Recombinant Proteins; D012965:Sodium Chloride
keywords: bacteriophage; ionization constant; nanoparticle; nuclear magnetic resonance (NMR); procapsid; protein folding; protein stability; virus