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Structure and dynamics of the DNA-binding protein HU of B. stearothermophilus investigated by Raman and ultraviolet-resonance Raman spectroscopy³

¹ Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA
² National and Kapodistrian University of Athens, Faculty of Biology, Department of Biochemistry and Molecular Biology, Panepistimiopolis-Zographou, 157 84 Athens, Greece

Reprint requests to: George J. Thomas, Jr., Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, USA; e-mail: thomasgj{at}umkc.edu; fax: (816) 235-1503.

The histone-like protein HU of Bacillus stearothermophilus (HUBst) is a 90-residue homodimer that binds nonspecifically to B DNA. Although the structure of the HUBst:DNA complex is not known, the proposed DNA-binding surface consists of extended arms that project from an -helical platform. Here, we report Raman and ultraviolet-resonance Raman (UVRR) spectra diagnostic of subunit secondary structures and indicative of key side-chains lining the proposed DNA-binding surface. Raman conformation markers show that the DNA-binding arms of the dimer contain ß-stranded structure in excess (eight ± two residues per subunit) of that reported previously. Important among side-chain markers are Met (701 cm^-1), Ala (908 cm^-1), Arg (1082 cm^-1), and Pro (1457 cm^-1). The Ala marker undergoes a substantial shift (908 893 cm^-1) on deuteration of alanyl peptide sites, indicating a coupled side-chain/main-chain mode of diagnostic value in the identification of exchange-protected alanines. A large subset of alanines (67%) in the -helical core exhibits robust resistance to exchange. A quantitative study of NH ND exchange exploiting newly identified amide II' markers of helical (1440 cm^-1) and nonhelical (1472 cm^-1) conformations of HUBst indicates unexpected flexibility at the dimer interface, which is manifested in rapid exchange of 80% of peptide sites. The results establish a basis for subsequent Raman and UVRR investigations of HUBst:DNA complexes and provide a framework for applications to other DNA-binding architectural proteins.

Keywords: Architectural protein; structure; conformation; DNA binding; DNA recognition; Raman spectroscopy; amide II'; hydrogen exchange

Abbreviations: CD, circular dichroism • CM, carboxymethyl • ds, double-stranded • EDTA, ethylenediamine tetraacetic acid • GuDCl, guanidinium deuteriochloride • GuHCl, guanidinium hydrochloride • H/D, hydrogen/deuterium • HU, histone-like prokaryotic DNA-binding protein • HUBst, protein HU from Bacillus stearothermophilus • IHF, integration host factor • UVRR, ultraviolet-resonance Raman • LB, Luria-Bertani • IPTG, isopropyl-ß-D-thiogalactoside; PLA, poly-L-arginine • PMSF, phenylmethylsulfonyl fluoride • SDS-PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis • Tris, tris(hydroxymethylamino)methane

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				K. Wojtuszewski and I. Mukerji The HU-DNA binding interaction probed with UV resonance Raman spectroscopy: Structural elements of specificity Protein Sci., September 1, 2004; 13(9): 2416 - 2428. [Abstract] [Full Text] [PDF]