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.
"We also found that the use of a small amount (10 ml per liter ) of 13 C– 15 N Bioexpress increased the yields U–[ 13 C]–[ 15 N]–KcsA to $40 mg/L of M9 using PASK90/JM83 system. This strategy that has been reported previously . A different strategy to increase the yield is to increase the concentration of the initial transfer of cells. "
[Show abstract][Hide abstract] ABSTRACT: We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of ∼35-40mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR.
Protein Expression and Purification 08/2013; 91(2). DOI:10.1016/j.pep.2013.07.013 · 1.70 Impact Factor
"Magic-angle spinning solid-state NMR (MAS SSNMR) represents a fast developing experimental method with great potential to provide structural and dynamics information for proteins not amenable to solution NMR nor X-ray crystallography. Many technical aspects of MAS SSNMR are rapidly developing, among them: (i) improvements in nano/microcrystalline and membrane protein sample preparation (Frericks et al. 2006; Li et al. 2007; Lorch et al. 2005) (ii) improvements in commercially available hardware, and (iii) development of pulse sequences for new and improved experiments (Sun et al. 1997; Li et al. 2007; Franks et al. 2007; Zhong et al. 2007; Hong 1999; Bockmann et al. 2003, Rienstra et al. 2000; Pauli et al. 2001; Igumenova et al. 2004; Astrof et al. 2001). In many cases, adaptation of tools and techniques from solution NMR have fueled this rapid development. "
[Show abstract][Hide abstract] 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.
"2D crystallization is a attractive option to achieve high protein concentrations for solid-state NMR. Higher concentrations have been achieved for example by preparing 3D crystals of the membrane protein DGK  or by precipitating the transmembrane enzyme DsbB . However, the best way to ensure a membrane environment at high protein concentration is 2D crystallisation, as demonstrated for the β-barrel outer-membrane porin OmpG . "
[Show abstract][Hide abstract] ABSTRACT: Proteins of the proteorhodopsin (PR) family are found abundantly in many marine bacteria in the photic zone of the oceans. They are colour-tuned to their environment. The green absorbing species has been shown to act as a light-driven proton pump and thus could form a potential source of energy. The pK(a) of the primary proton acceptor is close to the pH of seawater which could also indicate a regulatory role. Here, we review and summarize our own recent findings in the context of known data and present some new results. Proton transfer in vitro by PR is shown by a fluorescence assay which confirms a pH dependent vectoriality. Previously reported low diffracting 2D crystal preparations of PR are assessed for their use for solid-state NMR by two dimensional (13)C-(13)C DARR spectra. (15)N-(1)H HETCOR MAS NMR experiments show bound water in the vicinity of the protonated Schiff base which could play a role in proton transfer. The effect of highly conserved H75 onto the properties of the chromophore has been investigated by single site mutations. They do show a pronounced effect onto the optical absorption maximum and the pK(a) of the proton acceptor but have only a small effect onto the (15)N chemical shifts of the protonated Schiff base.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.