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.
"This was entirely unexpected since this toxin has a vastly different primary structure to ω-HXTX-Hv1a . 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 . "
[Show abstract][Hide abstract] ABSTRACT: Spiders have evolved pharmacologically complex venoms that serve to rapidly subdue prey and deter predators. The major toxic factors in most spider venoms are small, disulfide-rich peptides. While there is abundant evidence that snake venoms evolved by recruitment of genes encoding normal body proteins followed by extensive gene duplication accompanied by explosive structural and functional diversification, the evolutionary trajectory of spider-venom peptides is less clear.
Here we present evidence of a spider-toxin superfamily encoding a high degree of sequence and functional diversity that has evolved via accelerated duplication and diversification of a single ancestral gene. The peptides within this toxin superfamily are translated as prepropeptides that are posttranslationally processed to yield the mature toxin. The N-terminal signal sequence, as well as the protease recognition site at the junction of the propeptide and mature toxin are conserved, whereas the remainder of the propeptide and mature toxin sequences are variable. All toxin transcripts within this superfamily exhibit a striking cysteine codon bias. We show that different pharmacological classes of toxins within this peptide superfamily evolved under different evolutionary selection pressures.
Overall, this study reinforces the hypothesis that spiders use a combinatorial peptide library strategy to evolve a complex cocktail of peptide toxins that target neuronal receptors and ion channels in prey and predators. We show that the omega-hexatoxins that target insect voltage-gated calcium channels evolved under the influence of positive Darwinian selection in an episodic fashion, whereas the kappa-hexatoxins that target insect calcium-activated potassium channels appear to be under negative selection. A majority of the diversifying sites in the omega-hexatoxins are concentrated on the molecular surface of the toxins, thereby facilitating neofunctionalisation leading to new toxin pharmacology.
"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 , determining disulfide bond connectivity by NMR , engineering new structural motifs , 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 . "
[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.
[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.
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