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Hyberts, Sven

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Hyberts

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Sven

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Hyberts, Sven

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2014 - 20183

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Author's Publications: search.filters.isAuthorOfPublication.d7d8922e-87b7-4063-8045-b3c9679df006

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    Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression
    (American Chemical Society, 2014) Linser, Rasmus; Gelev, Vladimir; Hagn, Franz; Arthanari, Haribabu; Hyberts, Sven; Wagner, Gerhard
    Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins.
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    Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment
    (Nature Publishing Group UK, 2018) Robson, Scott; Takeuchi, Koh; Boeszoermenyi, Andras; Coote, Paul; Dubey, Abhinav; Hyberts, Sven; Wagner, Gerhard; Arthanari, Haribabu
    Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαβ) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment.
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    Solid-State NMR Structure Determination from Diagonal-Compensated, Sparsely Nonuniform-Sampled 4D Proton–Proton Restraints
    (American Chemical Society, 2014) Linser, Rasmus; Bardiaux, Benjamin; Andreas, Loren B.; Hyberts, Sven; Morris, Vanessa K.; Pintacuda, Guido; Sunde, Margaret; Kwan, Ann H.; Wagner, Gerhard
    We report acquisition of diagonal-compensated protein structural restraints from four-dimensional solid-state NMR spectra on extensively deuterated and 1H back-exchanged proteins. To achieve this, we use homonuclear 1H–1H correlations with diagonal suppression and nonuniform sampling (NUS). Suppression of the diagonal allows the accurate identification of cross-peaks which are otherwise obscured by the strong autocorrelation or whose intensity is biased due to partial overlap with the diagonal. The approach results in unambiguous spectral interpretation and relatively few but reliable restraints for structure calculation. In addition, the diagonal suppression produces a spectrum with low dynamic range for which ultrasparse NUS data sets can be readily reconstructed, allowing straightforward application of NUS with only 2% sampling density with the advantage of more heavily sampling time-domain regions of high signal intensity. The method is demonstrated here for two proteins, α-spectrin SH3 microcrystals and hydrophobin functional amyloids. For the case of SH3, suppression of the diagonal results in facilitated identification of unambiguous restraints and improvement of the quality of the calculated structural ensemble compared to nondiagonal-suppressed 4D spectra. For the only partly assigned hydrophobin rodlets, the structure is yet unknown. Applied to this protein of biological significance with large inhomogeneous broadening, the method allows identification of unambiguous crosspeaks that are otherwise obscured by the diagonal.