Fluorescence studies of the binding of bacteriophage M13 gene V mutant proteins to polynucleotides.
This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions. The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values. In addition, all proteins measured bind a more-or-less co-operative manner to single-stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H greater than wild-type greater than F68L and R16H greater than Y41F and Y41H greater than F73L greater than R21C greater than Y34H greater than G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.
Stassen
A P
AP
Laboratory of Molecular Biology, University of Nijmegen, The Netherlands.
Harmsen
B J
BJ
Schoenmakers
J G
JG
Hilbers
C W
CW
Konings
R N
RN
D001435:Bacteriophages / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005814:Genes, Viral; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011487:Protein Conformation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13