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].

Carboxypeptidase inhibitor

The Knottin PCI folds in a complex process

The folding pathway of the Potato Carboxypeptidase A Inhibitor PCI has been analyzed by structural analysis and stop/go folding experiments [Chang et al., 1994].
Folding of PCI proceeds through an initial stage of nonspecific disulfide formation.
1- and 2- and 3-disulfide intermediates are highly heterogeneous.
Disulfide reshuffling occurs at the final stage which refines and consolidates the scrambled species to acquire the native conformation.

Comparison with other small disulfide-rich proteins show that proteins such as PCI with their native disulfide bonds reduced in a collective and simultaneous manner exhibit both a high degree of heterogeneity of folding intermediates and the accumulation of scrambled isomers along the folding pathway [Chang & Bulychev, 2000a].

Analysis of the unfolding pathway of PCI has revealed the existence of structurally defined unfolding intermediates [Chang et al., 2000b]. It was also shown that the PCI sequence is unable to fold quantitatively into a single native structure, and that, under physiological conditions, the scrambled isomers of PCI that constitute about 4% of the protein are in equilibrium with native PCI.

PCI folding does not depend on the prosequence

Folding of the Potato Carboxypeptidase A Inhibitor PCI has been studied with or without the prosequences, either in vitro or in vivo in Escherichia coli [Bronsoms et al., 2003].
It is shown that the prosequence does not affect folding significantly neither in vitro nor in vivo.