EXPLORATION OF THE SINGLE-STRANDED DNA-BINDING DOMAINS OF THE GENE-V PROTEINS ENCODED BY THE FILAMENTOUS BACTERIOPHAGE-IKE AND BACTERIOPHAGE-M13 BY MEANS OF SPIN-LABELED OLIGONUCLEOTIDE AND LANTHANIDE-CHELATE COMPLEXES

Scrutiny of NOE data available for the protein encoded by gene V of th e filamentous phage IKe (IKe GVP), resulted in the elucidation of a be ta-sheet structure which is partly five stranded. The DNA-binding doma in of IKe GVP was investigated using a spin-labeled deoxytrinucleotide . The paramagnetic-relaxation effects observed in the H-1-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized us ing two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the st ructure of the protein. They exhibit a high degree of identity with re sidues in the gene V protein encoded by the distantly related phage M1 3 (M13 GVP), for which similar spectral perturbations are induced by s uch a spin-labeled oligonucleotide. Binding studies with negatively ch arged ayl-1,4,7,10-tetrakis(methylene)tetrakisphosphonic acid (DOTP) c omplexes, showed that these complexes bind to IKe and M13 GVP at two s patially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M1 3 GVP monomer, which coincides with the single-stranded DNA-binding do main as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phospha te binding) electropositive clusters at the surface of M13 and IKe GVP . These positive patches are interspersed with conserved or conservati vely replaced hydrophobic residues. At pH 5, a second Gd(DOTP)5--bindi ng site becomes apparent. The corresponding pattern of spectral pertur bations indicates the accommodation of patches of conserved, or conser vatively replaced, hydrophobic residues in the cores of the M13 and IK e dimers.