Partial B-to-A DNA Transition upon Minor Groove Binding of Protein Sac7d Monitored by Raman Spectroscopy † Lubomı ´r Dosta ´l, ‡ Chin-Yu Chen, §,| Andrew H.-J. Wang, § and Heinz Welfle* ,‡ AG Biopolymerspektroskopie, Max-Delbru ¨ck-Centrum fu ¨r Molekulare Medizin Berlin-Buch, Robert-Ro ¨ssle-Strae 10, D-13092 Berlin, Germany; Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Sec. 2, Nankang, Taipei 115, Taiwan; and Department of Chemistry, National Taiwan UniVersity, Taipei 106, Taiwan ReceiVed April 22, 2004; ReVised Manuscript ReceiVed May 27, 2004 ABSTRACT: Members of the Sso7d/Sac7d protein family and other related proteins are believed to play an important role in DNA packaging and maintenance in archeons. Sso7d/Sac7d are small, abundant, basic, and nonspecific DNA-binding proteins of the hyperthermophilic archeon Sulfolobus. Structures of several complexes of Sso7d/Sac7d with DNA octamers are known. These structures are characterized by sequence unspecific minor groove binding of the proteins and sharp kinking of the double helix. Corresponding Raman vibrational signatures have been identified in this study. A Raman spectroscopic analysis of Sac7d binding to the oligonucleotide decamer d(GAGGCGCCTC) 2 reveals large conformational perturbations in the DNA structure upon complex formation. Perturbed Raman bands are associated with the vibrational modes of the sugar phosphate backbone and frequency shifts of bands assigned to nucleoside vibrations. Large changes in the DNA backbone and partial B- to A-form DNA transitions are indicated that are closely associated with C2’-endo/anti to C3’-endo/anti conversion of the deoxyadenosyl moiety upon Sac7d binding. The major spectral feature of Sac7d binding is kinking of the DNA. Raman markers of minor groove binding do not largely contribute to spectral differences; however, clear indications for minor groove binding come from G-N2 and G-N3 signals that are supported by Trp24 features. Trp24 is the only tryptophan present in Sac7d and binds to guanine N3, as has been demonstrated clearly in X-ray structures of Sac7d-DNA complexes. No changes of the Sac7d secondary structure have been detected upon DNA binding. Complex formation with DNA-binding proteins drives DNA superhelicity, which in turn regulates the double helix stability. DNA in eubacteria and eukarya is overall negatively supercoiled due to gyrase action and wrapped around the histone core of the nucleosome. In contrast, episomal DNAs in thermophilic archaea are relaxed to positively supercoiled forms (1, 2). In archaea the mechanism for organizing DNA into a compact form is not well understood. Members of the Sso7d/Sac7d protein family and other related proteins are believed to play an important role in DNA packaging and maintenance. Sso7d/Sac7d are small, abundant, basic, and nonspecific DNA-binding proteins of the hyperthermo- philic archeon Sulfolobus (3, 4). Sso7d/Sac7d bind to DNA noncooperatively with micro- molar affinity, without strong sequence preference, and the T m values of Sac7-DNA complexes are ∼40 °C higher than those of free DNA (5). The structures of several Sac7d/ Sso7d-DNA complexes have been studied by X-ray crystal- lography (6-8), NMR (9), and low-angle X-ray scattering (10). Figure 1 shows a model of the Sac7d-DNA complex with octamer d(GCGATCGC) 2 (6). In the protein-DNA complexes, the protein structures are similar to those of the free proteins (3, 11), consisting of an incomplete -barrel made of a triple-stranded -sheet orthogonal to a -hairpin. The small -barrel is capped by an amphiphilic C-terminal R-helix. The triple-stranded -sheet is placed across the DNA minor groove, and the side chains of Val26 and Met29 intercalate into DNA base pairs † This work was supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg GK 80/1 to H.W.). * To whom correspondence should be addressed. E-mail: welfle@ mdc-berlin.de. Fax/Telephone: +49-30-9406 2840. Mailing address: Dr. Heinz Welfle, Max-Delbru ¨ck-Centrum fu ¨r Molekulare Medizin Berlin-Buch, Postfach 740238, D-13092 Berlin, Germany. ‡ Max-Delbru ¨ck-Centrum fu ¨r Molekulare Medizin Berlin-Buch. § Academia Sinica. | National Taiwan University. FIGURE 1: Model of the Sac7d-d(GCGATCGC) 2 complex. The figure was prepared using SPDBV (DeepView/Swiss-PdbViewer from http://www.expasy.org/spdb/) and the atomic coordinates from the pdb file 1AZP. 9600 Biochemistry 2004, 43, 9600-9609 10.1021/bi049192r CCC: $27.50 © 2004 American Chemical Society Published on Web 07/09/2004