Chemical shift assignment of the transmembrane helices of DsbB, a 20‐kDa integral membrane enzyme, by 3D magic‐angle spinning NMR spectroscopy

Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Protein Science (Impact Factor: 2.85). 03/2008; 17(2):199-204. DOI: 10.1110/ps.073225008
Source: PubMed


The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential 13C and 15N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by three-dimensional (3D) correlation experiments on a uniformly 13C, 15N-labeled sample at 750-MHz 1H frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.

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Available from: Ying Li, Oct 08, 2015
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    • "Protocols for expression, refolding, and purification of recombinantly expressed human VDAC were based on the work of Malia and Wagner(Malia and Wagner 2007) and Hiller et al(Hiller et al. 2008b). 2D crystals were prepared according to the procedures published in Eddy et al.(Eddy et al. 2012), modified from the protocol originally described by Dolder et al.(Dolder et al. 1999). "
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    ABSTRACT: The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for 13C line widths and <0.5 ppm 15N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via 1H detected MAS NMR.
    Journal of Biomolecular NMR 01/2015; 61(3-4). DOI:10.1007/s10858-015-9903-1 · 3.14 Impact Factor
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    • "Magic angle spinning (MAS) nuclear magnetic resonance (NMR) is an effective method to extract biologically relevant structural constraints with sub-angstrom precision from a variety of different systems such as proteins embedded in native lipid bilayers [1] [2] [3] [4] [5] [6] [7] [8], antibiotics bound to bacterial cell walls in whole cells [9] and amyloid fibrils [10] [11] [12] [13] [14]. All three of these are examples of systems that are not accessible by either X-ray crystallography or solution NMR spectroscopy, the standard tools of structural biology. "
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    ABSTRACT: We present a calculation of the microwave field distribution in a magic angle spinning (MAS) probe utilized in dynamic nuclear polarization (DNP) experiments. The microwave magnetic field (B(1S)) profile was obtained from simulations performed with the High Frequency Structure Simulator (HFSS) software suite, using a model that includes the launching antenna, the outer Kel-F stator housing coated with Ag, the RF coil, and the 4mm diameter sapphire rotor containing the sample. The predicted average B(1S) field is 13μT/W(1/2), where S denotes the electron spin. For a routinely achievable input power of 5W the corresponding value is γ(S)B(1S)=0.84MHz. The calculations provide insights into the coupling of the microwave power to the sample, including reflections from the RF coil and diffraction of the power transmitted through the coil. The variation of enhancement with rotor wall thickness was also successfully simulated. A second, simplified calculation was performed using a single pass model based on Gaussian beam propagation and Fresnel diffraction. This model provided additional physical insight and was in good agreement with the full HFSS simulation. These calculations indicate approaches to increasing the coupling of the microwave power to the sample, including the use of a converging lens and fine adjustment of the spacing of the windings of the RF coil. The present results should prove useful in optimizing the coupling of microwave power to the sample in future DNP experiments. Finally, the results of the simulation were used to predict the cross effect DNP enhancement (ϵ) vs. ω(1S)/(2π) for a sample of (13)C-urea dissolved in a 60:40 glycerol/water mixture containing the polarizing agent TOTAPOL; very good agreement was obtained between theory and experiment.
    Journal of Magnetic Resonance 05/2011; 210(1):16-23. DOI:10.1016/j.jmr.2011.02.001 · 2.51 Impact Factor
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    • "Labs have published assignment results using category I strategies, but only on small proteins (Hong 1999; Pauli et al. 2001; Igumenova et al. 2004; Franks et al. 2005; Balayssac et al. 2007). Labs are starting to use category II strategies for larger proteins (Frericks et al. 2006; Li et al. 2007; Li et al. 2008). It is expected that labs in the future will probably explore category III strategies using newer G-matrix Fourier transformation (GFT) experiments(Szyperski et al. 1993a; Szyperski et al. 1993b; Kim and Szyperski 2003; Kim and Szyperski 2004; Astrof et al. 2001; Luca and Baldus 2002). "
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    ABSTRACT: Magic-angle spinning solid-state NMR (MAS SSNMR) represents a fast developing experimental technique with great potential to provide structural and dynamics information for proteins not amenable to other methods. However, few automated analysis tools are currently available for MAS SSNMR. We present a methodology for automating protein resonance assignments of MAS SSNMR spectral data and its application to experimental peak lists of the β1 immunoglobulin binding domain of protein G (GB1) derived from a uniformly 13C- and 15N-labeled sample. This application to the 56 amino acid GB1 produced an overall 84.1% assignment of the N, CO, CA, and CB resonances with no errors using peak lists from NCACX 3D, CANcoCA 3D, and CANCOCX 4D experiments. This proof of concept demonstrates the tractability of this problem. Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9448-2) contains supplementary material, which is available to authorized users.
    Journal of Biomolecular NMR 10/2010; 48(3):123-8. DOI:10.1007/s10858-010-9448-2 · 3.14 Impact Factor
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