1H, 13C, and 15N resonance assignments and secondary structure analysis of the HU protein from Bacillus stearothermophilus using two- and three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy.
Nearly complete 1H, 13C, and 15N resonance assignments have been obtained for the protein HU from Bacillus stearothermophilus (dimer, 19.5 kDa) using double- and triple-resonance 2D and 3D NMR experiments. This has resulted in assignments of 91% of the observable protons, 98% of all 13C, and 92% of all 15N nuclei. NOEs obtained from a 3D time-shared NOESY-(13C,15N)-HSQC spectrum, exchange data of amide protons, and chemical shifts of the 1H alpha, 1HN, 13C beta, 13C alpha, 13CO, and 15N nuclei have been used to identify the secondary structure elements. Three alpha-helices (residues 3-13, 18-37, and 83-90) and three extended strands (residues 40-45, 48-62, and 67-82) have been found in HU. The arrangement of these elements of secondary structure is very similar to the X-ray structure [Tanaka et al. (1984) Nature 310, 376-381; White et al. (1989) Proteins 5, 281-288]. The conformation of the proposed DNA-binding region of HU, i.e., an antiparallel beta-hairpin, was not observed previously in the X-ray structure. In the NMR structure long range NOEs in the beta-arm region (residues 53-76) suggest a distortion between residue Pro-72 and Ala-73 and between Pro-63 and Gln-64 with concomitant distortions in the opposite strand. The NOE data indicate further that the loop region in the DNA-binding arms of HU is arranged as a type I beta-turn from Pro-63 to Gly-66.
Vis
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Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
Boelens
R
R
Mariani
M
M
Stroop
R
R
Vorgias
C E
CE
Wilson
K S
KS
Kaptein
R
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D004268:DNA-Binding Proteins / Q000737:chemistry*; D001411:Geobacillus stearothermophilus / Q000737:chemistry*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D017433:Protein Structure, Secondary
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D009587:Nitrogen Isotopes; C040330:histone-like protein HU, bacteria; D006859:Hydrogen