Article

Partial13C and15N Chemical-Shift Assignments of the Disulfide-Bond-Forming Enzyme DsbB by 3D Magic-Angle Spinning NMR Spectroscopy

Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
ChemBioChem (Impact Factor: 3.06). 03/2007; 8(4):434-42. DOI: 10.1002/cbic.200600484
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
84 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ferritins are intracellular proteins that can store thousands of iron(III) ions as a solid mineral. These structures autoassemble from four-helix bundle subunits to form a hollow sphere and are a prototypical example of protein nanocages. The protein acts as a reservoir, encapsulating iron as ferric oxide in its central cavity in a nontoxic and bioavailable form. Scientists have long known the structural details of the protein shell, owing to very high resolution X-ray structures of the apoform. However, the atomic level mechanism governing the multistep biomineralization process remained largely elusive. Through analysis of the chemical behavior of ferritin mutants, chemists have found the role of some residues in key reaction steps. Using Mössbauer and XAS, they have identified some di-iron intermediates of the catalytic reaction trapped by rapid freeze quench. However, structural information about the iron interaction sites remains scarce. The entire process is governed by a number of specific, but weak, interactions between the protein shell and the iron species moving across the cage. While this situation may constitute a major problem for crystallography, NMR spectroscopy represents an optimal tool to detect and characterize transient species involving soluble proteins. Regardless, NMR analysis of the 480 kDa ferritin represents a real challenge. Our interest in ferritin chemistry inspired us to use an original combination of solution and solid state approaches. While the highly symmetric structure of the homo-24-mer frog ferritin greatly simplifies the spectra, the large protein size hinders the efficient coherence transfer in solution, thus preventing the sequence specific assignments. In contrast, extensive (13)C-spin diffusion makes the solution (13)C-(13)C NOESY experiment our gold standard to monitor protein side chains both in the apoprotein alone and in its interaction with paramagnetic iron species, inducing line broadening on the resonances of nearby residues. We could retrieve the structural information embedded in the (13)C-(13)C NOESY due to a partial sequence specific assignment of protein backbone and side chains we obtained from solid state MAS NMR of ferritin microcrystals. We used the 59 assigned amino acids (∼33% of the total) as probes to locate paramagnetic ferric species in the protein cage. Through this approach, we could identify ferric dimers at the ferroxidase site and on their pathway towards the nanocage. Comparison with existing data on bacterioferritins and bacterial ferritins, as well as with eukaryotic ferritins loaded with various nonfunctional divalent ions, allowed us to reinterpret the available information. The resulting picture of the ferroxidase site is slightly different with various ferritins but is designed to provide multiple and generally weak iron ligands. The latter assist binding of two incoming iron(II) ions in two proximal positions to facilitate coupling with oxygen. Subsequent oxidation is accompanied by a decrease in the metal-metal distance (consistent with XAS/Mössbauer) and in the number of protein residues involved in metal coordination, facilitating the release of products as di-iron clusters under the effect of new incoming iron(II) ions.
    Accounts of Chemical Research 09/2013; 46(11). DOI:10.1021/ar4000983 · 24.35 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical-shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein-protein and protein-lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane.
    Journal of Molecular Biology 02/2014; DOI:10.1016/j.jmb.2014.02.007 · 3.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Double cross polarization (DCP) has been widely used for heteronuclear polarization transfer between (13)C and (15)N in solid-state magic-angle spinning (MAS) NMR. However, DCP is such sensitive to experimental settings that small variations or deviations in RF fields would deteriorate its efficiency. Here, we report on asymmetric simultaneous phase-inversion cross polarization (referred as aSPICP) for selective polarization transfer between low-γ (13)C and (15)N spins. We have demonstrated through simulations and experiments using biological solids that the asymmetric duration in the simultaneous phase-inversion cross polarization scheme leads to efficient polarization transfer between (13)C and (15)N even with large chemical shift anisotropies in the presence of B1 field variations or mismatch of the Hartmann-Hahn conditions. This could be very useful in the aspect of long-duration experiments for membrane protein studies at high fields.
    Journal of Magnetic Resonance 03/2014; 242C:214-219. DOI:10.1016/j.jmr.2014.03.002 · 2.32 Impact Factor