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


Spider toxins


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

α-Amylase Inhibitor

Oxydative folding of AAI involves intermediates with nonnative disulfide bridges between adjacent cycteines

The folding pathway of the α-Amylase Inhibitor AAI from Amaranthus has been analyzed using RP-HPLC, LC-MS, 1H-NMR and photochemically induced dynamic nuclear polarization (photo-CIDNP) experiments [Cemazar et al., 2003].
Folding of AAI proceeds through several fully oxydized intermediates with nonnative disulfide bridges.
The major folding intermediate (MFI) is shown to contain a disulfide bridge between adjacent cysteine residues, i.e. Cys60 and Cys61 according to the knottin unique numbering.

A clear interdependence between the formation of disulfide bridges and conformational folding is demonstrated [Cemazar et al., 2004]. It is proposed that formation of nonnative disulfide bridges facilitates folding by reducing the entropy of the unfolded state. Then the conversion from nonnative disulfide bridges to native disulfide bridges is driven by the formation of native stabilizing non-bonding interactions.