Viable transmembrane region mutants of bacteriophage M13 coat protein prepared by site-directed mutagenesis.
Bacteriophage M13 coat protein - a 50-residue protein located at the E. coli host membrane during phage reproduction - is subjected to cytoplasmic, membrane-bound, and DNA-interactive environments during the phage life cycle. In research to examine the specific features of primary/secondary structure in the effective transmembrane (TM) region of the protein (residues 21-39: YIGYAWAMVVVIVGATIGI) which modulate its capacity to respond conformationally to the progressive influences of these varying environments, we have prepared over two dozen viable mutant phages with alterations in their coat protein TM regions. Mutants were obtained through use of site-directed mutagenesis techniques in combination with three "randomized" oligonucleotides which spanned the TM region. No subcloning was required. Among mutations observed were those in which each of the four TM Val residues was changed to Ala, and several with increased Ser or Thr content, including one double Ser mutant (G23S-A25S). Polar substitutions arising at Gly23 and Tyr24-including G23D, Y24H, Y24D and Y24N-suggested that this local segment resides external to the host membrane. Milligram quantities of mutant coat proteins are obtained by growing M13 mutant phages in liter preparations, with isotopic (e.g., 13C) labelling at desired sites, for subsequent characterization and conformational analysis in membrane-mimetic media.
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Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Deber
C M
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D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D036022:Capsid Proteins; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D019746:Intramolecular Oxidoreductases; D007535:Isomerases; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D036022:Capsid Proteins; D011994:Recombinant Proteins; C025668:coat protein, Bacteriophage M13; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase