Site-directed mutagenesis of the M13 gene 5 protein: the role of Arg-21, Tyr-26 and Tyr-41.
The gene 5 protein of bacteriophage M13 is a single stranded DNA binding protein essential for phage replication. We have generated the mutations R21A, Y26F and Y41A in the gene 5 protein and purified the mutant proteins for functional characterisation in vitro. The complex of Y26F with single-stranded DNA is disrupted at 0.8 M NaCl, the same salt concentration as that required to dissociate the native complex. However, the mutant proteins R21A and Y41A are considerably less stable and dissociate from single-stranded DNA at at 0.4 M NaCl. The fluorescence of the mutant proteins and the DNA-protein complexes they form has been compared with the wild-type protein to allow an assessment of the contribution from individual residues. We conclude that the fluorescence of Tyr-26 is 50% quenched in the complex with DNA, whereas that of Tyr-41 is fully quenched. Fluorescence titrations of the mutant proteins with poly(dT) show that all three mutant proteins can bind DNA but, in the case of Y41A, with a change of stoichiometry suggesting a loss of cooperativity. Gel retardation analysis of Y41A also shows anomalous behaviour in binding to oligonucleotides, consistent with the proposed involvement of Tyr-41 in dimer-dimer contacts in the nucleoprotein complex.
Turner
G P
GP
Biophysics Laboratories, University of Portsmouth, UK.
Kneale
G G
GG
D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13; D014443:Tyrosine; D001120:Arginine