In this study, we assess the effects of substituting a diselenide for a native disulfide cross-link in the well-characterized inhibitory cystine knot peptide, ω-conotoxin GVIA. A diselenide was substituted for each of the three native disulfides and the resulting selenocysteine-containing analogs were evaluated for whether the substitution affected either oxidative folding or/and function. The cross-linking pattern of each folded analog of ω-conotoxin GVIA (containing a diselenide and two disulfides) was determined by NMR.
ω-Conotoxin GVIA is a well-characterized pore blocker of voltage-gated calcium channels with high affinity for the CaV
2.2 subtype (31
). Thus, one apparent advantage of using ω-GVIA as a model for testing the diselenide substitution was an easy and sensitive assessment of this approach on the bioactivity of the ICK-motif containing peptide. Electrophysiological experiments using HEK293 cells expressing the CaV
2.2 channel demonstrated that all three Sec analogs of ω-conotoxin GVIA are fully functional compared to native ω-conotoxin GVIA. The in vivo
functional activity of the Sec-GVIA analogs was not detectably different from ω-GVIA. Such benign effects of disulfide-to-diselenide were previously observed in other Conus
peptides, namely α- and µ-selenoconopeptides targeting nicotinic acetylcholine receptors and voltage-gated sodium channels, respectively (15
). These examples add to a growing list of bioactive peptides containing diselenide bridges (62
) and encourage further efforts to explore the selenopeptide strategy for discovery efforts and structure/function studies of disulfide-rich peptides (63
Marked differences with respect to oxidative folding between Sec-GVIA analogs and ω-GVIA were observed. For all three Sec analogs, the kinetics of folding were significantly faster, and the yields greater. The best yield was obtained when the second disulfide linkage (Cys8–Cys19) was replaced by a diselenide (an increase to 78%). The effects of diselenide bridges on folding kinetics were striking; it took significantly longer to fold ω-GVIA (t1/2 > 50 minutes) than any of the Sec-GVIA analogs (t1/2 < 10 minutes). Thus, substituting a diselenide for a native disulfide linkage in GVIA had no detectable effect on function but a marked acceleration in the kinetics of folding, and an increase in yield of the properly folded analog. Our proof-of-concept findings with Sec-GVIA are encouraging to apply the diselenide replacement to more difficult to chemically synthesize and oxidized ICK peptides, such as µO- or δ-conotoxins.
Thermodynamic and kinetic studies on Sec-GVIA analogs emphasize the importance of loop sizes of disulfides/diselenides in increasing the yield and rate of accumulation of the natively folded peptides. The larger the size of a preformed diselenide loop, the faster the rate of accumulation of natively folded peptide. Effectiveness of improving the folding yield by site-specific incorporation of selenocysteine may depend on the formation of non-native disulfides, which also depends on the loop sizes of all the remaining possible disulfide connectivities (three distinct combinations exist for forming two disulfide bridges). The possibility of forming small disulfide loops in GVIA[C1U,C16U] and GVIA[C15U,C26U] resulted in the accumulation of non-natively folded peptides, decreasing the yield of forming the natively folded species. Comparable and larger size of non-native disulfide loops in GVIA[C8U,C19U] may have precluded the accumulation of the non-natively folded species, thus increasing the overall folding yield. These findings suggest how to design analogs of cysteine-rich peptides with site-specific incorporation of selenocysteine to rationally engineer the oxidative folding pathways. More studies are clearly needed to generalize these findings to other than the ICK disulfide scaffolds.
Our results also support broader use of selectively-labeled 13
N cysteine residues for disulfide mapping by means of NMR. In general, the following rules may assist preferential incorporation of labeled cysteine in disulfide rich peptides and subsequent efficient disulfide mapping. (1
) Cysteines with well-dispersed Hβ
chemical shifts should be first selected for labeling as they potentially can provide the largest number of cross-disulfide NOEs. (2
) In case of degenerate Hβ
chemical shifts, intra-residue degenerate Hβ
s should be preferred over inter-residue degenerate Hβ
s for labeling. High-resolution NMR spectroscopy together with careful selection for incorporation of 13
N labeled cysteine residues in disulfide rich peptides should assist rapid disulfide mapping. Recently, 77
Se NMR was used to determine a diselenide connectivity in a selenopeptide analog of spider toxin κ-ACTX-Hv1c (64
), further confirming that NMR-based methods may offer an attractive alternative to a traditional mapping of disulfide bridges by partial reduction, followed by a proteolytic or chemical fragmentation and mass spectrometry analysis.
The results demonstrate that diselenide substitution for a native disulfide bond may be a feasible strategy for producing ICK peptide analogs that retain the biological activity of the native peptide, but have improved folding kinetics and yields. The improved folding kinetics could be particularly beneficial in those cases where ICK peptides fold very inefficiently and tend to aggregate during the folding reaction. These are all candidates for using the diselenide strategy. We are presently determining whether a dramatic improvement in yield can be attained in these cases. The ICK peptides comprise structurally and functionally diverse group of bioactive peptides, including neurotoxins, protease inhibitors, antiviral cyclotides and antimicrobial peptides. Research on ICK peptides involves drug discovery and preclinical/clinical development, validating this group of peptides as a rich source of new, potential first-in-class, biotherapeutics. Our work encourages using the integrated oxidative folding approach to study structure and function of the ICK peptides.