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

摘要

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 α-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 β-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 α-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.