Exploration of the single-stranded DNA-binding domains of the gene V proteins encoded by the filamentous bacteriophages IKe and M13 by means of spin-labeled oligonucleotide and lanthanide-chelate complexes.
Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.
Nijmegen SON Research Centre for Molecular Design, Structure and Synthesis, University of Nijmegen, The Netherlands.
J P
JP
Van Duynhoven
I M
IM
Nooren
D W
DW
Swinkels
P J
PJ
Folkers
B J
BJ
Harmsen
R N
RN
Konings
G I
GI
Tesser
C W
CW
Hilbers
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics*; D001665:Binding Sites; D002614:Chelating Agents / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008674:Metals, Rare Earth / Q000737:chemistry*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry; D013113:Spin Labels; D014764:Viral Proteins / Q000378:metabolism*
D002614:Chelating Agents; D004277:DNA, Single-Stranded; D008674:Metals, Rare Earth; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D013113:Spin Labels; D014764:Viral Proteins; C068293:gene 5 protein, Enterobacteria phage T4; C082084:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonic acid)