Conformational states of mutant M13 coat proteins are regulated by transmembrane residues.
Mutational and structural analysis of the 28 viable bacteriophage M13 mutants obtained by randomized mutagenesis of the effective transmembrane (TM) segment of the 50-residue major coat (gene VIII) protein (residues 21-39) demonstrated that M13 coat protein functionality, as reflected by phage viability, is incompatible with an increase in Gly + beta-branched residue content in its TM core. SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy performed in membrane environments on purified mutant coat proteins revealed that these proteins exist in a range of state(s), identified as helical monomers and dimers and polymeric (alpha-helical and/or beta-sheet) species, of which relative populations, and thermally induced conformational transitions, were dependent uniquely upon mutation type and locus. Mutations to relatively polar residues (e.g. G23D, Y24D, Y24H, A27E, I32T, and T36S) stabilized principally monomeric species, while mutants with decreased beta-branched content in the protein TM hydrophobic core (e.g. V29A, V30A, V31A, V31L, and V33A) displayed mainly dimeric species. Mutation of Ile37-->Thr within a "Sternberg-Gullick" consensus sequence of the coat protein TM segment led to a highly oligomerized/polymerized protein. The overall results suggest that TM residues in M13 coat protein are not universal components of a hydrophobic anchor segment per se, but are further selected (i) to impart conformational flexibility to the TM segment through helix destabilization and (ii) to retain the capacity to regulate protein-protein association and packing motifs within membranes.
Li
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Division of Biochemistry Research, Hospital for Sick Children, Toronto, Ontario, Canada.
Glibowicka
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Joensson
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Deber
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D036022:Capsid Proteins; D002462:Cell Membrane / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation
D036022:Capsid Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; C025668:coat protein, Bacteriophage M13