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.

The 60-Residue C-Terminal Region of the Single-Stranded DNA Binding Protein of Herpes Simplex Virus Type 1 Is Required for Cooperative DNA Binding

“…Both in the presence and in the absence of ssDNA, ICP8 was digested in less than 3 min into two fragments of approximately 95 and 33 kDa. However, in the presence of (dT) 20 , the 95-kDa polypeptide was more resistant to further cleavage than the unligated species. The cleavage pattern is in agreement with previous trypsin digestion studies (72,73) implicating the 56-kDa fragment as the smallest product containing the ssDNA binding domain.…”

“…At the same time, we wished to identify possible deletions that would reduce the tendency of the protein to aggregate. The sensitivity to trypsin cleavage of the full-length ICP8 alone or in complex with (dT) 20 was analyzed in time course experiments. As shown in Fig.…”

“…Loading buffer (90% formamide, 10% 10ϫ Trisborate-EDTA [TBE] buffer, 0.025% bromophenol blue, 0.025% xylene cyanol, 20% glycerol) was added at 1:10; then the samples were heated for 5 min at 95°C, chilled for 5 min on ice, and spun in an Eppendorf table centrifuge prior to application to a TBE denaturing gel supplemented with 8 M urea. The acrylamide-bisacrylamide (19:1) concentration was adjusted to 12% for (dT) 28 and (dT) 35 or to 15% for the shorter (dT) 14 and (dT) 20 . The gel was run at 40 V/cm.…”

“…About 48 g of wild-type ICP8 was digested with 0.7 g of trypsin in 90 l of a binding buffer consisting of 0.1 M HEPES (pH 7.5), 150 mM NaCl, and 2 mM MgCl 2 . Stock solutions of (dT) 20 oligonucleotides were dissolved in the same binding buffer. To detect any DNA protection from proteolytic cleavage, the same amount of protein was incubated with a 15-fold molar excess of (dT) 20 for 30 min on ice prior to digestion with trypsin.…”

“…Stock solutions of (dT) 20 oligonucleotides were dissolved in the same binding buffer. To detect any DNA protection from proteolytic cleavage, the same amount of protein was incubated with a 15-fold molar excess of (dT) 20 for 30 min on ice prior to digestion with trypsin. At the times indicated, 2 g of protein was removed from the mixture, and proteolysis was terminated by boiling the sample for 5 min in loading buffer.…”