Structure and dynamics of the DNA-binding protein HU of B. stearothermophilus investigated by Raman and ultraviolet-resonance Raman spectroscopy.
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 alpha-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 beta-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 alpha-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.
Serban
Doinita
D
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA.
Arcineigas
Sandra F
SF
Vorgias
Constantinos E
CE
Thomas
George J
GJ
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000378:metabolism; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C040330:histone-like protein HU, bacteria; D001120:Arginine; D011392:Proline; D000409:Alanine