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


Spider toxins


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

Squash inhibitors

The Knottins EETI-II and MCoTI-II fold in a simple process

The folding process of the squash inhibitor EETI-II has been studied in several steps

Folding of EETI-II is fast and efficient. Air oxidation of cysteines leads in few hours and with good yield to native disulfide bridges [Le-Nguyen et al., 1989b]

A linear synthetic analog of EETI-II in which all cysteines have been replaced by serines, has been obtained and submitted to careful NMR analyses [Heitz et al., 1995]. Native local structure propensities are clearly detected for the 11-15 310-helix region and for the 22-25 β-turn region. Only very few native non-covalent interactions are detected (proximity between Phe26 and residues Val20)

Mono-disulfide synthetic analogs of EETI-II in which cysteines 2, 9, 19 and 21 or 2, 19, 15 and 27 were replaced by serines, have been synthesized. These compounds do not display more native-like 3D structurations than does the linear analog analog [Heitz et al., 1997]. These results indicate that formation of the first disulfide bridge is non-specific and that at least two disulfides are necessary to stabilize the native fold.

A stable two-disulfide intermediate in the folding process displays most 3D structure of the native compound [Le-Nguyen et al, 1993]. It is shown that the two disulfides II-V and III-VI are necessary AND sufficient to determine the native fold. This corresponds to the elementary two-disulfide Cystine-Stabilized β-sheet (CSB) motif. Similar results have been reported for kalata B1 (see Cyclotides).

The folding pathway of the macrocyclic squash inhibitor MCoTI-II was found to be very similar to that of the linear EETI-II (see above). This study was performed on the natural cyclized compound although it is unknown if in vivo the head-to-tail cyclization occurs prior to oxidative folding. It is likely that oxydative folding of the linear compound precedes cyclization [Cemazar et al., 2006; Cemazar et al., 2008]

The GPNG 22-25 β-turn in EETI-II favors folding and is sequence restricted

The 22-25 GPNG β-turn appeared as the most flexible part in native EETI-II [Chiche et al., 1989; Chiche et al., 1993]. Nevertheless, replacing the corresponding sequence in the homologous inhibitor CMTI-III by the GPNG sequence strongly improved the chemical synthesis yield of the latter compound [Rolka et al., 1991b].

Supporting this observation, a combinatorial library of EETI-II in which the 22-25 GPNG sequence was randomized revealed that the sequence of this turn is significantly constrained and that only the native GPNG sequence provides high yield in the native fold [Wentzel et al., 1999].