EXPLORING THE DNA-BINDING DOMAIN OF GENE-V PROTEIN ENCODED BY BACTERIOPHAGE M13 WITH THE AID OF SPIN-LABELED OLIGONUCLEOTIDES IN COMBINATION WITH H-1-NMR

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of H-1 nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the H-1-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.