Connectivity Conotoxins

Scheme 10 Disulfide Connectivities of Isomers of >Conotoxin GIIIB x i...

All the disulfide-folding pathways have the common feature that disulfide formation is initially random and not energetically favored due to the unfolded state of the reduced polypeptide chain. Disulfide formation becomes less random as certain disulfides are favored energetically as a result of the acquisition of nonrandom local conformations. In turn, the disulfides stabilize such conformations, so that disulfide formation and folding become cooperative. Thereby the sequence encoded structural information may fully suffice to direct the correct oxidative refolding as shown for co-conotoxin GVIA (9) (Section 6.1.5.2.1) which assumes the native-type cystine connectivities directly even in absence of redox reagents and... 

Scheme 11 Disulfide Connectivities of Isomers of co-Conotoxin MVIICJ89 ...

Scheme 6 Formation of Two Interchain Disulfide Bridges with Different Connectivities Using Two Kinds of Protecting Groups for the Cysteine Residues. Synthetic Schemes for the Two Isomers of the Tryptic/Chymo-tryptic Fragment of (0-Conotoxin-GVIA,62l...

Table 1 Eight Possible Lysyl Endopeptidase Fragments of p-Conotoxin GIIIB with Different Disulfide Connectivities and Ratios of (PTH-Cys)2 during Their Sequencing 72 "...

The controversy over the connectivity does highlight the fact that the determination of disulphide connectivities in disulphide-rich peptides is notoriously difficult and caution needs to be exercised in using solely NMR methods to determine disulphide connectivity. Isotopic labelling approaches116 have been recently used for conotoxins, a disulphide-rich class of animal toxins that are described in more detail later in this article. 

The majority of conotoxin structures (115 of 125 structures in the PDB) have been determined by NMR spectroscopy as opposed to X-ray crystallography. They have significant structural diversity as a result of their diverse sequences and disulphide connectivities. Furthermore, conotoxins also have a large number of post-translational modifications, including the presence of hydroxyproline, y-carboxyglutamic acid and bromotryptophan residues, epimerisation, glycosyla-tion and amidation,144,145 that enhance their structural diversity. A recent analysis of NMR data for the unusual amino acids present in conotoxins3 provides a reference source for chemical shifts of post-translationally modified amino acids. 

There is an intimate relationship between the conopeptide type, its structure, and its selectivity different types of conopeptides target different neuronal receptor types. However, with the same conopeptide type (or subtypes), selectivity varies tremendously, as different sites within the same receptor can be targeted by the conopeptides whose difference is only one amino acid. This is illustrated by the selectivity of a-conotoxins (Table 3) toward the nAChR. This is further augmented by a new level of conotoxin diversity, as a nonnative disulfide bond connectivity in a-conotoxin AuIB reduces structural definition but increases biological activity. ... 

In cocultures of fetal neurons from ventral half of the spinal cord (VH neurons) and from the dorsal root ganglion (DRG neurons) the synaptic transmission between pairs of spinal cord neurons from ventral half of the spinal cord (VH-VH connections) or between DRG neurons and VH neurons (DGR and VH connections) were studied with two cell recording and stimulation techniques. In 70% of the VH-VH connections and in 50% of the DGR-VH connections, Bay K 8644 failed to affect transmitter release. co-Conotoxin GVIA produced no consistent effect on EPSPs or IPSPs elicited by VH neurons by stimulation of the nearby neurons. VH EPSPs elicited by stimulation of the nearby DGR neurons were reduced by 50% by co-couotoxiu GVIA. Therefore, ueither sustaiued nor inactivating HVA Ca channels sensitive to Bay K 8644 or co-conotoxin GVIA such as those measured in the neuronal cell body are responsible for action-potential-evoked transmitter release from the majority of the VH neurons these channels may be involved in transmitter release in approximately 30% of these neurons [152]. 

Valentijn, K. et al., Omega-conotoxin- and nifedipine-insensitive voltage-operated calcium channels mediate K(-t)-induced release of pro-thyrotropin-releasing hormone-connecting peptides Ps4 and Ps5 from perifused rat hypothalamic slices. Brain Res Mol Brain Res 14, 221, 1992. 

With the exception of a-conotoxin SII from Conus striatus, all a-conotoxins have the cysteine pattern, CC—C—C (Myers et al., 1993) (see Table 4). Peptides from the fish-hunting species, C geographus, C. striatus and C. magus, have the consensus core sequence CC(N/H)PACGXX(Y/F)XC and two disulfide bonds that connect Cys to Cys and Cys to Cys. Comparison with the a-conotoxins recently isolated from C. pennaceus, a mollusc-hunter, and C. imperialism a worm-hunter, indicates variations in the size of the intercysteine loops. The second loop has seven amino acid residues in a-conotoxins PnIA and PnIB from C pennaceus (Fainzilber et al., 1994), and only three residues in a-conotoxin Iml from C. imperialis (McIntosh et al., 1994), compared to five residues in the other a-conotoxins. 

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