12538 zyxwvutsrqpo Biochemistry zyxwvu 1993,32, 12538-12547 Characterization of the Pf3 Single-Strand DNA Binding Protein by Circular Dichroism Spectroscopy+ Michael D. Powell and Donald M. Gray' Program in Molecular and Cell Biology, Mail Stop FO 31, The University of Texas at Dallas, P.O. Box 830688, Richardson, Texos 75083-0688 Received April I, 1993; Revised Manuscript Received August 16, 1993% ABSTRACT: We have used circular dichroism (CD) spectroscopy and gel electrophoresis to characterize the single-strand DNA binding protein (ssDBP) of the bacteriophage Pf3 and its complexes with Pf3 DNA and various DNA and RNA homopolymers. The secondary structure of Pf3 ssDBP had zyxw < 1 % a-helix and therefore was probably a @-sheet structure like the fd gene zyxwv 5 protein (g5p). From CD titrations, the binding stoichiometry of Pf3 ssDBP was two nucleotides per protein monomer (n = 2) for complexes formed with all of the nucleic acids except poly[r(U)], for which n = 3 (in a buffer of 10 mM Tris-HC1 and 70 mM NaC1, pH 8.2). Evidence of an additional binding mode of n = 4 for complexes formed with Pf3 DNA was found by gel electrophoresis experiments. Pf3 ssDBP showed a marked sequence dependence in binding affinities similar to that known for the fd g5p. The gene 5 protein (g5p) * of bacteriophage Ff (M13,f 1 ,fd) is a model for the single-strand DNA @DNA) binding proteins of filamentous phages (Kowalczykowski et al., 1981). The fd g5p plays both structural and regulatory roles during the phage life cycle. Alberts et al. (1972) showed that the g5p binds tightly and cooperatively to ssDNA in a stoichiometric manner (one protein monomer per every four DNA nucle- otides, and the binding is salt-sensitive). In addition, the binding of g5p to ssDNA results in unstacking of the DNA bases (Alberts et al., 1972). Pretorius et al. (1975) and Cavalieri et al. (1976) have shown by sedimentation equi- librium that the fd g5p exists primarily as a dimer in solution. The helix-destabilizing propertiesof this protein help it promote the change from the synthesis of double-stranded replicative form DNA to the synthesis of single-stranded viral genome DNA (Oey & Knippers, 1972; Salstrom L Pratt, 1971). The fd g5p has been shown to be a translationalrepressor of multiple phage proteins (Zaman et al., 1991), and the protein plays a structural role in the prepackagingof viral DNA in preparation for the extrusion of mature phage. All of these functions are carried out by a protein of only 87 amino acids. The crystal structure of the fd g5p without bound nucleic acid has been solved by X-ray diffraction (Brayer & McPherson, 1983;Skinneret al., 1993). Thesolutionstructure has been investigated by IH NMR (de Jong et al., 1987,1989; King & Coleman, 1988; Folkers et al., 1991) and other techniques, such as circular dichroism (CD) spectroscopy, linear dichroism (LD) spectroscopy, electron spin resonance (ESR), and fluorescence spectroscopy [van Amerongen et al., 1990; see Kansy et al. (1986) for a review]. fd g5p is predominantly a @sheet structure arranged into three major loops: a complex loop, a dyad loop, and a DNA binding loop. Recently, the DNA binding loop has been refined by 2D NMR This work was performed by M.D.P. in partial fulfillment of the requirements for the Ph.D. degree in the Program in Molecular and Cell Biology, The University of Texas at Dallas. Support was provided by NIH Research Grant GM 19060 and by Grant AT-503 from the Robert A. Welch Foundation. * Abstract published in Advance ACS Abstracts, November 1, 1993. Abbreviations: ssDBP,singlestrand DNA bindingprotein,g5p, gene 5 protein; Tris, tris(hydroxymethy1)aminomethane; CD, circular dichro- ism; OD, optical density; EDTA, ethylenediaminetetraacetic acid; TAE buffer, Tris-acetate (0.04 M) and EDTA (0.001 M), pH 8.0. 0006-2960/93/0432-12538$04.00/0 studies and has been shown to be present in IKe g5p, wild- type M13, and a Y41H mutant of M13 (van Duynhoven et al., 1990;Folkers et al., 1991). Mutational analysesarebeing carried out on fd g5p in an effort to better understand the underlying protein-nucleic acid interactions (Stassen et al., 1992; Zabin et al., 1991). One important result from these studies is that there appear to be different residues involved in the specific binding to the 5'-untranslated region of the gene 11 mRNA and in the nonspecific binding to ssDNA. The filamentous phage Pf3 is of a different structural class than the Ff phage (Day et al., 1988) and has Pseudomonas ueruginosu (RP1) asits host (Stanisich, 1974). Thenucleotide sequence of Pf3 has been determined, and although it shares little overall sequence homology to the Ff phage, there is an apparent conservation of gene arrangement and relative size and numbers of open reading frames (Luiten et al., 1985). The coding regions for ssDNA binding proteins have been identified in both Pf3 (78 amino acids, 8907 Da; Putterman et al., 1984) and fd (87 amino acids, 9690 Da; Cuypers et al., 1974). A comparison of the amino acid sequences of fd g5p and the ssDNA binding protein (ssDBP) of Pf3 reveals little overall homology. There does, however, appear to be con- servation of particular amino acids, especially in the region of the DNA binding loop of fd g5p (Peeters et al., 1983). The in vivo precursor complex of Pf3 has been isolated and studied by electron microscopy, UV absorbance, CD spec- troscopy, and sedimentation analysis (Casadevall & Day, 1985). In Casadevall and Day's study, the in uiuo complex of Pf3 appeared to adopt a helical conformation similar to that seen with the fd g5p complex. In the current study, we have utilized purified protein to reconstitute complexes in vitro. The properties of these complexes should provide a valuable comparison with those formed by the fd g5p and aid in further elucidation of the general chemical and structural bases of the mechanisms for this entire class of proteins. CD spectroscopy is particularly suited to the study of the aromatic residues of these proteins and the conformational changes of nucleic acids upon binding. Neither fd g5p nor Pf3 ssDBP has tryptophan residues, so that tyrosines provide the major source of optical activity in the 220-240-nm region. Of the five tyrosines present in fd g5p, three (Tyr-26, Tyr-56, 0 1993 American Chemical Society