Role of aromatic residues at the lipid-water interface in micelle-bound bacteriophage M13 major coat protein.
Analyses of transmembrane domains of proteins have revealed that aromatic residues tend to cluster at or near the lipid-water interface of the membrane. To assess protein-membrane interactions of such residues, a viable mutant library was generated of the major coat protein of bacteriophage M13 (a model single membrane-spanning protein) in which one or the other of its interfacial tyrosine residues (Tyr-21 and Tyr-24) is mutated. Using the interfacial tryptophan (Trp-26) as an intrinsic probe, blue shifts in fluorescence emission spectra and quenching constants indicated that mutants with a polar amino acid substitution (such as Y24D or Y24N) are less buried in a deoxycholate micelle environment than in the wild type protein. These polar mutants also exhibited alpha-helix to beta-structure transition temperatures in incremental-heating circular dichroism studies relatively lower than those of wild type and nonpolar mutants (such as Y21V, Y21I, and Y24A), indicating that specific side chains in the lipid-water interface influence local protein-micelle interactions. Mutant Y21F exhibited the highest transition temperature, suggesting that phenylalanine is ostensibly the most effective interfacial anchoring residue. Using phage viability as the assay in a combination of site-directed and saturation mutagenesis experiments, it was further observed that both Tyr residues could not simultaneously be "knocked out". The overall results support the notion that an interfacial Tyr is a primary recognition element for precise strand positioning in vivo, a function that apparently cannot be performed optimally by residues with simple aliphatic character.
Yuen
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Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
Davidson
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Deber
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017104:Bacteriophage M13 / Q000737:chemistry* / Q000235:genetics; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D036022:Capsid Proteins; D003840:Deoxycholic Acid / Q000737:chemistry; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008823:Micelles; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics; D014867:Water / Q000737:chemistry*
D036022:Capsid Proteins; D003902:Detergents; D008563:Membrane Lipids; D008565:Membrane Proteins; D008823:Micelles; C025668:coat protein, Bacteriophage M13; D003840:Deoxycholic Acid; D014867:Water; D014443:Tyrosine; D014364:Tryptophan