REFINED STRUCTURE, DNA-BINDING STUDIES, AND DYNAMICS OF THE BACTERIOPHAGE-PF3 ENCODED SINGLE-STRANDED-DNA BINDING-PROTEIN

The solution structure of the 18-kDa single-stranded DNA binding prote in encoded by the filamentous Pseudomonas bacteriophage Pf3 has been r efined using 40 ms N-15- and C-13-edited NOESY spectra and many homo- and heteronuclear J-couplings. The structures are highly precise, but some variation was found in the orientation of the beta-hairpin denote d the DNA binding wing with respect to the core of the protein. Backbo ne dynamics of the protein was investigated in the presence and absenc e of DNA by measuring the R-1 and R-2 relaxation rates of the N-15 nuc lei and the N-15-H-1 NOE. It was found that the DNA binding wing is mu ch more flexible than the rest of the protein, but its mobility is lar gely arrested upon binding of the protein to d(A)(6). This confirms ea rlier hypotheses on the role of this hairpin in the function of the pr otein, as will be discussed. Furthermore, the complete DNA binding dom ain of the protein has been mapped by recording two-dimensional TOCSY spectra of the protein in the presence and absence of a small amount o f spin-labeled oligonucleotide. The roles of specific residues in DNA binding were assessed by stoichiometric titration of d(A)(6), which in dicated for instance that Phe43 forms base stacking interactions with the single-stranded DNA. Finally, all results were combined to form a set of experimental restraints, which were subsequently used in restra ined molecular dynamics calculations aimed at building a model for the Pf3 nucleoprotein complex. Implying in addition some similarities to the well-studied M13 complex, a plausible model could be constructed t hat is in accordance with the experimental data.