Mutagenesis of bacteriophage IKe major coat protein transmembrane domain: role of an interfacial proline residue.
The transmembrane (TM) domain of the 53-residue major coat protein of the M13-related bacteriophage IKe (residues 24-42: LISQTWPVVTTVVVAGVLI) has been subjected to randomized mutagenesis to probe the conformation and stability of the TM domain, as well as the effect of structurally-important residues such as proline. TM mutants were obtained by the Eckstein method of site-directed mutagenesis using the IKe genome as template so as to eliminate the need for subcloning. Over 40 single- and double-site viable mutants of bacteriophage IKe were isolated. Every residue in the TM segment, except the highly conserved Trp29, could be mutated to at least one other residue; polar and charged mutations occurred in the TM segment adjacent to the N-terminal domain (residues 24-28), while non-polar substitutions predominated in the C-terminal portion (residues 30-42). The Pro30 locus tolerated four mutations-Ala, Gly, Cys, and Ser- which represent the four side chains of least volume. Mutant coat proteins obtained directly from the phage in milligram quantities were studied by circular dichroism spectroscopy and SDS-PAGE gels. Wild type IKe coat protein solubilized in sodium deoxycholate micelles was found to occur as an alpha-helical, monomeric species which is stable at 95 degrees C, whereas the mutant Pro30-->Gly undergoes an irreversible conformational transition at ca. 90 degrees C to an aggregated beta-sheet structure. The result that Pro30 stabilizes the TM helix in the micellar membrane suggests a sterically-restricted location for the wild type Pro pyrrolidine side chain in the bulky Trp-Pro-Val triad, where it may be positioned to direct the initiation of the subsequent TM core domain helix.
Williams
K A
KA
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Deber
C M
CM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D004252:DNA Mutational Analysis; D017912:Inovirus / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics*; D017433:Protein Structure, Secondary
D008565:Membrane Proteins; D011392:Proline