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 r esidues tend to cluster at or near the lipid-water interface of the membran e. To assess protein-membrane interactions of such residues, a viable mutan t library was generated of the major coat protein of bacteriophage M13 (a m odel single membrane-spanning protein) in which one or the other of its int erfacial tyrosine residues (Tyr-21 and Tyr-24) is mutated. Using the interf acial tryptophan (Trp-26) as an intrinsic probe, blue shifts in fluorescenc e emission spectra and quenching constants indicated that mutants with a po lar amino acid substitution (such as Y24D or Y24N) are less buried in a deo xycholate micelle environment than in the wild type protein. These polar mu tants also exhibited cl-helix to p-structure transition temperatures in inc remental-heating circular dichroism studies relatively lower than those of wild type and nonpolar mutants (such as Y21V, Y21I, and Y24A), indicating t hat specific side chains in the lipid-water interface influence local prote in-micelle interactions. Mutant Y21F exhibited the highest transition tempe rature, suggesting that phenylalanine is ostensibly the most effective inte rfacial anchoring residue. Using phage viability as the assay in a combinat ion 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 primar y recognition element for precise strand positioning in vivo, a function th at apparently cannot be performed optimally by residues with simple aliphat ic character.