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].

Cyclotides

Reviews on the oxidative folding of cyclotides are available [Cemazar et al., 2008; Gunasekera et al., 2009]


In vivo Cyclization of cyclotides probably involves an asparaginyl endopeptidase

A highly conserved Asn residue is present at the C-terminus of the mature sequence of most cyclotides, suggesting that an Asn specific enzyme could be involved in the in vivo cyclization. When Oak1, the precursor of kalata B1, is expressed in Nicotiana Benthamiana, a plant which contains well characterized asparaginyl endopeptidases, cyclic oxidized kalata B1 is produced. Interestingly, inhibition or silencing of the asparaginyl endopeptidases in Nicotiana Benthamiana did not affect the production of kalata B1 but significantly decreased the production of the cyclic compound [Saska et al., 2007].

Cyclization favors correct folding of Kalata B1

Oxidation of cysteines during chemical synthesis of Kalata B1 has been performed using two different strategies [Daly et al., 1999]. It is shown that oxidation of the linear precursor, before cyclisation, necessitate an hydrophobic environment. In contrast, if cyclization is performed prior to oxidation, then the hydrphobic environment is unnecessary. This highlights the favorable role of the cyclization in the folding of Kalata B1.

A two-disulfide intermediate accumulates during folding of Kalata B1

Examination of oxidative refolding of kalata B1 shows that a native-like intermediate containing two out of the three native disulfide bridges is accumulated [Daly et al., 2003]. This intermediate contains the II-V and III-VI disufide bridges and is analogous to the intermediate previously observed during folding of EETI-II. In contrast to EETI-II, however, this kalata B1 folding intermediate is a kinetic trap and is not the immediate precursor of the native form.