Different DNA-binding modes and cooperativities for bacteriophage M13 gene-5 protein revealed by means of fluorescence depolarisation studies.
The binding of the bacteriophage-M13-encoded gene-5 protein to oligo(deoxythymidylic acid)s and M13 DNA was studied by means of tyrosyl fluorescence decay and fluorescence anisotropy measurements. The observed fluorescence decays could be described with two exponentials, characterised by the lifetimes tau 1 = 2.2 ns and tau 2 = 0.8 ns respectively. Only the amplitude of the longer-lifetime component is influenced by binding of the protein to DNA. This indicates that a part of the tyrosyl residues is involved in the binding. By means of fluorescence depolarisation measurements the rotational correlation time of the protein dimer is found to be 12.9 ns. In contrast to earlier measurements, carried out on the DNA-binding protein of phage Pf1 [Kneale, G. G. and Wijnaendts van Resandt, R. W. (1985) Eur. J. Biochem. 149, 85-93], the observed rotational correlation times of the gene-5 protein pass through a maximum when the protein is titrated with oligo(deoxythymidylic acid)s. This is not observed upon titration with M13 DNA. Our measurements showed that for the oligo(deoxythymidylic acid)s there clearly is a decrease in the number of clustered proteins on the lattice in the case of excess nucleotide. This is a direct consequence of the much lower cooperativity of the binding to the oligonucleotides compared to the cooperativity characteristic of binding to polynucleotides. The number of nucleotides covered by a protein monomer is found to be less than or equal to 3 for the oligonucleotides and approximately equal to 4 for M13 DNA. Model calculations show that the 'time-window' through which the fluorescence depolarisation can be observed (i.e. the fluorescence lifetime) in this case significantly affects the 'measured' effective rotational correlation times.
D003090:Coliphages / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005454:Fluorescence Polarization; D008433:Mathematics; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins