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The HU–DNA binding interaction probed with UV resonance Raman spectroscopy: Structural elements of specificity

Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459-0175, USA

(RECEIVED March 9, 2004; FINAL REVISION May 7, 2004; ACCEPTED May 18, 2004)

The Escherichia coli protein HU functions as an architectural DNA-binding protein by facilitating DNA looping or bending to form multiprotein complexes. Although HU does not recognize a specific DNA sequence, site-specific binding to a number of discontinuous, looped, or bent DNA substrates has been observed. In this study UV resonance Raman (UVRR) spectroscopy is used to identify structural elements associated with low- and high-affinity binding by examining three different HU–DNA complexes. UVRR spectra obtained with an excitation wavelength of 210 nm, which preferentially enhances protein backbone amide vibrations, indicate that HU secondary structure content increases and the protein structure becomes more rigid upon binding to DNA. The increase in -helical content is attributed to the C-terminal helix, which interacts with the DNA and may play a role in binding affinity and specificity. UVRR spectra obtained with a 215 nm excitation wavelength demonstrate that Pro mode intensity at 1455 cm^–1 decreases upon complex formation. This intensity decrease is attributed to the intercalation of Pro residues between DNA base pairs to induce a bend in the DNA, as has been observed previously in the IHF–DNA and HU–DNA cocrystal structures. DNA vibrational modes are also indicative of significant base unstacking and opening of the minor groove upon protein binding, consistent with bending and distortion of the DNA. In all three complexes, A-DNA conformational features are indicated by deoxyribose-phosphate backbone modes. These and other results suggest that protein-induced bending plays an important role in HU site-specific binding and supports a model of a mutually induced fit.

Keywords: protein; DNA interactions; A-tracts; Raman spectroscopy; DNA bending; DNA binding

Abbreviations: bp, base pair • IHF, integration host factor • FRET, fluorescence resonance energy transfer • UVRR, UV resonance Raman spectroscopy.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04730204.

Reprint requests to: Ishita Mukerji, Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, Middletown, CT 06459-0175, USA; e-mail: imukerji{at}wesleyan.edu; fax: (860) 685-2141.

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