Partial (13)C and (15)N chemical-shift assignments of the disulfide-bond-forming enzyme DsbB by 3D magic-angle spinning NMR spectroscopy.
DsbB is a 20 kDa Escherichia coli inner-membrane protein that catalyzes disulfide-bond formation in periplasmic proteins. We report highly resolved, multidimensional magic-angle spinning NMR spectra at 750 MHz (1)H frequency, which enable partial (13)C and (15)N chemical-shift assignments of the signals. The narrow line widths observed indicate excellent microscopic order of the protein sample, suitable for full structure determination by solid-state NMR. Experiments were performed exclusively on uniformly (13)C,(15)N-labeled DsbB. Chemical-shift-correlation experiments based on dipolar transfer yielded strong signals in the 3D spectra, many of which have been site-specifically assigned to the four transmembrane helices of DsbB. Significant numbers of additional residues have been assigned to stretches of amino acids, although not yet placed in the amino acid sequence. We also report the temperature dependence of signal intensities from -50 degrees C to 0 degrees C, a range over which samples of DsbB are highly stable. Structural and dynamic information derived from SSNMR studies can give insight into DsbB in a state that so far has not been successfully crystallized.
Li
Ying
Y
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Berthold
Deborah A
DA
Frericks
Heather L
HL
Gennis
Robert B
RB
Rienstra
Chad M
CM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D001426:Bacterial Proteins; D002247:Carbon Isotopes; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009587:Nitrogen Isotopes