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 in a more‐or‐less co‐operative manner to single‐stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H > wild‐type > F68L and R16H > Y41F and Y41H > F73L > R21C > Y34H > 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 homopoly‐nucleotides is largely non‐specific.