title: Refined structure, DNA binding studies, and dynamics of the bacteriophage Pf3 encoded single-stranded DNA binding protein.
abstract: The solution structure of the 18-kDa single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been refined using 40 ms 15N- and 13C-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 denoted the DNA binding wing with respect to the core of the protein. Backbone dynamics of the protein was investigated in the presence and absence of DNA by measuring the R1 and R2 relaxation rates of the 15N nuclei and the 15N-1H NOE. It was found that the DNA binding wing is much more flexible than the rest of the protein, but its mobility is largely arrested upon binding of the protein to d(A)6. This confirms earlier hypotheses on the role of this hairpin in the function of the protein, as will be discussed. Furthermore, the complete DNA binding domain of the protein has been mapped by recording two-dimensional TOCSY spectra of the protein in the presence and absence of a small amount of spin-labeled oligonucleotide. The roles of specific residues in DNA binding were assessed by stoichiometric titration of d(A)6, which indicated 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 restrained 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 that is in accordance with the experimental data.
authors.affiliation: Nijmegen SON Research Center, Laboratory of Biophysical Chemistry, University of Nijmegen, The Netherlands.
authors.forename: R H
authors.initials: RH
authors.lastname: Folmer
authors.forename: M
authors.initials: M
authors.lastname: Nilges
authors.forename: C H
authors.initials: CH
authors.lastname: Papavoine
authors.forename: B J
authors.initials: BJ
authors.lastname: Harmsen
authors.forename: R N
authors.initials: RN
authors.lastname: Konings
authors.forename: C W
authors.initials: CW
authors.lastname: Hilbers
mesh_terms: D018360:Crystallography, X-Ray; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009698:Nucleoproteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017105:Pseudomonas Phages / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014764:Viral Proteins