Direct Visualization of Disulfide Bonds through Diselenide Proxies Using Se-77 NMR Spectroscopy

Institute for Molecular Bioscience, The University of Queensland, St. Lucia QLD 4072, Australia.
Angewandte Chemie International Edition (Impact Factor: 11.26). 11/2009; 48(49):9312-4. DOI: 10.1002/anie.200905206
Source: PubMed


Se-ing is believing: Many proteins are cross-braced by disulfide bonds that frequently play key roles in protein structure, folding, and function. Unfortunately, the methods available for assignment of disulfide-bond connectivities in proteins are technically difficult and prone to misinterpretation. Now disulfide bond connectivities in native proteins can be visualized directly using 77Se NMR spectroscopy.

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    • "This was entirely unexpected since this toxin has a vastly different primary structure to ω-HXTX-Hv1a [39]. Moreover, in addition to the six conserved cysteine residues in ω-HXTX-Hv1a that form an inhibitor cystine knot (ICK) motif [40,41], κ-HXTX-Hv1c contains two additional cysteine residues that form an extremely rare vicinal disulfide bond [42-44]. Furthermore, in contrast to ω-HXTX-Hv1a, which blocks insect CaV channels, κ-HXTX-Hv1c is a potent and specific blocker of KCa channels [45]. "
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    BMC Genomics 03/2014; 15(1):177. DOI:10.1186/1471-2164-15-177 · 3.99 Impact Factor
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    • "Subsequent evolution of O-ribosomes enabled the selection of ribosomes that enhanced the incorporation of unnatural amino acids at UAG stop codons (b), the decoding of quadruplet codons (c), and the incorporation of selenocysteine at UGA codons flanked by a selenocysteine insertion sequence (SECIS) in the mRNA (d). with an increased capacity to incorporate selenocysteine could be useful for many synthetic biology and biotechnological applications . For example, high-level incorporation of selenocysteine into recombinant proteins will facilitate phasing in X-ray crystallography [75], determining disulfide bond connectivity by NMR [76], engineering new structural motifs [77], and the characterization and re-engineering of naturally occurring selenoproteins. Furthermore , results using suppressor tRNAs emphasize that selenocysteine incorporation is a distinct process from canonical nonsense suppression of translation termination [72]. "
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    FEBS letters 02/2013; 587(8). DOI:10.1016/j.febslet.2013.02.032 · 3.17 Impact Factor
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    ABSTRACT: Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.
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