title: Biophysical characterization of wild-type and mutant bacteriophage IKe major coat protein in the virion and in detergent micelles.
abstract: Interactions between the filamentous bacteriophage major coat protein and its environment differ markedly between the membrane-bound assembly intermediate which spans the lipid bilayer and the phage coat protein which makes up the capsid of the virion. Nonetheless, both reflect successful strategies to sequester the hydrophobic regions of the coat protein away from the aqueous milieu. To characterize the roles of individual residues in the conformation, stability, and oligomerization of the coat protein in both the virion and in detergent micelles, wild-type IKe and M13 coat proteins, together with a library of over 40 IKe coat protein mutants, were studied using circular dichroism (CD), fluorescence, and solution nuclear magnetic resonance (NMR) spectroscopies. The largely helical conformations of coat protein in IKe wild-type and mutant virions were found to be very similar by CD, demonstrating that the overall organization of the phage can accommodate a diverse range of amino acid substitutions in the major coat protein. Intrinsic Trp fluorescence showed that the polarity of the Trp 29 environment in the virion was modulated by residues within one helical turn of this locus. Characterization of IKe phage growth and plaquing properties highlighted the importance of Pro 30 in maintaining viability. As well, the Pro 30 mutants were the only substitutions which rendered the detergent-solubilized coat protein less thermostable and additionally altered the polarity of the Trp 29 environment. The Pro 30 Gly mutant exhibited numerous 1H and 15N chemical shift changes between residues Ile 25 and Ala 38 in the 2D 1H-15N HSQC spectrum in myristoyllysophosphatidylglycerol (MPG) micelles, demonstrating that the effect of the substitution is propagated beyond adjacent residues. The overall results highlight the stabilizing effect of Pro in the first turn of a transmembrane helix and the importance of hydrophobicity in modulating the oligomerization and stability of coat protein both in the phage and in detergent micelles.
authors: Division of Biochemistry Research, Hospital for Sick Children, Toronto, Ontario, Canada.
authors: K A
authors: KA
authors: Williams
authors: C M
authors: CM
authors: Deber
mesh_terms: D000595:Amino Acid Sequence; D001704:Biopolymers; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D036022:Capsid Proteins; D002942:Circular Dichroism; D003090:Coliphages / Q000737:chemistry / Q000254:growth & development; D003902:Detergents; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D014771:Virion / Q000737:chemistry*
chemical_list: D001704:Biopolymers; D036022:Capsid Proteins; D003902:Detergents; D008823:Micelles; C099133:major coat protein, bacteriophage IKe