Direct Visualization of Disulfide Bonds through Diselenide Proxies Using Se-77 NMR Spectroscopy
ABSTRACT 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.
- SourceAvailable from: Oliver Rackham[Show abstract] [Hide abstract]
ABSTRACT: It was previously thought that the proteins produced by ribosomes were dictated only by the sequences of the mRNAs they translated, however now it is apparent that subpopulations of ribosomes can have unique properties that influence the functions of the proteins they produce. Ribosomes have been engineered to discriminate between different mRNA templates or with unique decoding properties, and many new applications of unnatural ribosomes can be foreseen. In natural systems ribosomes with alternate protein and RNA composition have been shown to selectively translate specific mRNAs. As more is learned about ribosome structure and the mechanisms of translation, new opportunities to engineer ribosomes for applications in biotechnology and synthetic biology can be developed and new examples of ribosome-mediated regulation of translation are likely to emerge in nature.FEBS letters 02/2013; 587(8). DOI:10.1016/j.febslet.2013.02.032 · 3.34 Impact Factor
- [Show abstract] [Hide abstract]
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.Biochemistry 02/2010; 49(12):2741-52. DOI:10.1021/bi902137c · 3.19 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged μ-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of another disulfide bridge with a diselenide bridge) dramatically simplified its oxidative folding while preserving the peptide’s ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogues containing a single disulfide bridge allowed rapid positional scanning at Lys7 of μ-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide’s selectivity in blocking a neuronal (Nav1.2) over a muscle (Nav1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high-throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.Keywords (keywords): Conotoxins; diselenide bridges; selenocysteines; oxidative folding; disulfide-rich peptidesACS Medicinal Chemistry Letters 05/2010; 1(4). DOI:10.1021/ml900017q · 3.07 Impact Factor