The KNOTTIN database

Folding may imply complex equilibrium and disulfide reshuffling

  •  Although disulfide bridges are responsible for the high stability of Knottins, they also render the folding process more complex, especially in chemical synthesis.
  • Since Knottins are considered as interesting leads in drug design, it is essential to understand the basic principles that govern the folding process. This would help in rational knottin-based drug-design studies.
  • Main historical and recent efforts along this way are outlined below.
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Squash inhibitors

alpha-Amylase Inhibitor

Carboxypeptidase inhibitor

Conotoxins

Spider toxins

Cyclotides

General review on the oxidative folding of small disulfide-rich proteins are available [Arolas et al, 2006; Craik 2010].

Conotoxins

A review on the oxidative folding of conotoxins is available [Bulaj & Olivera, 2008]


Peptidyl prolyl cis-trans isomerases facilitate conotoxin folding

Peptidyl prolyl cis-trans isomerases isolated from the venom gland of Conus novaehollandiae were shown to greatly increase the rate of appearance of hydoxyproline containing conotoxins [Safavi-Hemami et al., 2010].

Hydroxyprolines facilitates folding of ω-conotoxins but do not modify the acitivy

It has been shown that hydroxylation of Prolines improved the in vitro folding yield of ω-MVIIC approximately 2-fold. Conotoxin ω-GVIA displayed similar results. In contrast, hydroxylation of non knottin μ-conotoxins affected activity rather than folding [Lopez-Vera et al., 2008].

Albumin is a redox-active crowding agent that promotes oxidative folding of conotoxins and other cysteine-rich peptides

Oxidative folding reactions were shown to be dramatically accelerated when protein-based crowding agents were present at concentrations lower than those predicted to provide the excluded volume effect. Submillimolar albumin alone appeared as effective as glutathione in promoting the oxidative folding of GI conotoxin [Buczek et al., 2007].

The pro-peptide might play a role in the PDI-catalized folding of conotoxins

The influence of the propeptide on the folding of α-conotoxins has been analyzed [Buczek et al., 2004]. It is shown that the propeptide does not significantly affect oxidative folding of α-conotoxin GI, it does facilitate PDI-assisted folding (PDI: protein disulfide isomerase). It is proposed that the increased length of the precursor may favor binding to PDI. Although α-conotoxins do not share the knottin fold, this result might be of importance for folding of other disulfide-rich proteins.

Non-ionic detergents and low temperature improve folding

Factors that improve oxidative folding of hydrophobic δ-conotoxins have been searched [DeLa Cruz et al., 2003]. It is shown that non-ionic detergents at low temperature (0°C) significantly improves the folding yield of δ-conotoxin PVIA. This effect is attributed to stabilization of native PVIA through non-specific interactions between the hydrophobic part of the detergent and non-polar patches at the surface of PVIA.

Two disufides are necessary for native 3D interaction to occur in omega-conotoxin MVIIA

Three omega-conotoxin MVIIA analogs in which the cysteines of one of the three disulfide bridges are replaced by alanines have been synthesized [Price-Carter et al., 2002; Price-Carter et al., 1998]. For each analog, all mono and two disulfide-bonded species were identified and equilibrium constants evaluated. These studies indicate that, in contrast with results for EETI-II, two-disulfide compounds are not significantly stabilized by non-covalent interactions. However, formation of two native disulfide greatly favor formation of the third disulfide and the native folded structure.

Mature conotoxins are able to refold in vitro

It is shown that in presence of GSSG/GSH, several conotoxins are able to refold to their native conformation with efficiencies ranging from 16% (omega-MVIIC) to 50% (omega-MVIIA, -GVIA and SVIA) [Price-Carter et al., 1996a; Price-Carter et al., 1996b]. Effect of salt and temperature on omega-MVIIC native disulfide brige formation has been reported [Kubo et al., 1996]

The Cys15-Cys26 disulfide is essential for correct folding of omega-conotoxin GVIA

The Cys15-Cys26 disulfide bridge in omega-conotoxin GVIA has been deleted by replacing corresponding cysteines by serines [Flinn et al., 1999]. This analog displayed a gross loss of secondary and tertiary structure, probably responsible for the total absence of activity.