Most of the small peptides in scorpion venom are surprisingly robust for their size. The main source of their resistance to heat, pH changes and degrading enzymes is the disulfide bridges that link different points of the chain together. This article looks at the chemistry of the bridge, its role in peptide structure and how it is studied in the laboratory.
What is a disulfide bridge?
Cysteine, one of the twenty standard amino acids, carries a thiol group in its side chain, that is, an end made up of sulfur and hydrogen. When two cysteine residues come close enough in space, the hydrogens of the thiol groups are removed by an oxidation reaction and a covalent bond forms between the two sulfur atoms. The resulting sulfur-sulfur bond is called a disulfide bridge, and the pair of cysteines linked by it is called cystine.
A bridge can join two points that are far apart in the chain. Thanks to these links, a linear amino acid sequence is locked into a particular three-dimensional shape. Hydrogen bonds and hydrophobic interactions also support folding, but these are weak and easily reversed interactions; the disulfide bridge, being covalent, is much stronger.
In the cell, bridges do not form at random. Proteins destined for secretion fold in a special compartment within the cell, in an oxidizing environment, and helper enzymes that break incorrectly formed bonds and re-pair them take part in this process. The cells of the venom gland prepare their peptides in the same way and release them into the secretion in folded form.
Disulfide bridges are not unique to venoms. Hormones such as insulin, antibodies and many defence peptides also stabilize their structure with these bonds; the peptides in spider and cone snail venoms are likewise rich in cysteine. A high number of bridges is a common feature of molecules that are mostly secreted outside the cell and must function in harsh environments.
The bridge pattern in scorpion toxins
A large proportion of scorpion toxins share a common scaffold known as the cysteine-stabilized alpha-beta fold. In this structure, a short helical region is linked by disulfide bridges to a two- or three-stranded beta sheet. There are three residues between the two cysteines on the helix and a single residue between the two cysteines on the beta strand; this pattern is one of the sequence signatures used to recognize the family.
| Peptide group | Typical length | Typical number of bridges |
|---|---|---|
| Potassium channel toxins (α-KTx) | Approximately 23–43 amino acids | 3, in some members 4 |
| Sodium channel toxins | Approximately 60–76 amino acids | 4 |
| Chlorotoxin | 36 amino acids | 4 |
The structure and targets of the long-chain group were covered in the article on sodium channel toxins.
A reading based on the peptide in the reference batch
The 36-amino-acid sequence of the α-KTx 3.6 peptide identified in the reference batch report is FTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS. The sequence contains six cysteines, located, counting from the start, at positions 6, 12, 16, 27, 32 and 34. There are three residues between the cysteines at positions 12 and 16 and a single residue between those at positions 32 and 34; in other words, the signature described above can be seen in this sequence as well.
In these six-cysteine peptides, the pairing pattern most often reported in the literature links the first cysteine with the fourth, the second with the fifth and the third with the sixth. However, counting the cysteines in a sequence does not prove which cysteine is paired with which; the actual pairing can only be shown by the experimental methods described below. The reference batch report gives no measurement result on bridge pairing, so no assumed pattern is stated here. The scope of the report is set out on the analysis page.
How do the bridges provide robustness?
An unfolded peptide chain can adopt countless shapes; the folded state represents only one of these possibilities. Disulfide bridges limit from the outset the number of shapes into which the chain can freely open up. Moving away from the folded structure thus becomes energetically more difficult, and the molecule retains its shape for longer when the temperature rises or ambient conditions change.
Resistance to enzymes stems from the same compactness. Proteases need a flexible, exposed section of the chain in order to reach the bond they cut; a small peptide tightly packed by bridges has few such regions. This property makes it easier for the peptides to serve as usable research tools and makes them relatively robust in storage, whether dry or in solution.
The weak point of the bridges
The bridges are strong but not unbreakable. Reducing agents open the sulfur-sulfur bond and return the cysteines to free thiols. In the laboratory, compounds such as dithiothreitol, beta-mercaptoethanol or TCEP are used for this purpose. In protein analysis this is a deliberate step: reduction carried out before SDS-PAGE opens the bridges and changes the molecule's behaviour in the gel.
The second risk is disulfide exchange. When free thiol groups are present and the pH rises, existing bridges can swap with free thiols and mispaired structures can form; under acidic conditions this exchange slows down. Chemically capping free cysteines during analysis, that is, alkylating them, is a common way of preventing this scrambling.
How are the bridges studied?
- Mass measurement: Two hydrogen atoms are removed when each bridge forms, so the folded peptide is about 2 daltons lighter per bridge than its reduced form. For a peptide with three bridges, the difference is about 6 daltons.
- Reduction comparison: Reduced and non-reduced forms of the same sample are run side by side in chromatography or electrophoresis.
- Digestion without reduction: The peptide is cleaved enzymatically with the bridges kept intact; the fragments that remain linked by a bridge show which cysteines are paired.
- Structure determination: Nuclear magnetic resonance and crystallography show the three-dimensional positions of the bridges directly.
Mass differences are measured precisely using the method described in the article on mass spectrometry.
When the quantity obtained from natural sources is insufficient, chemical synthesis or recombinant production is used. The linear chain obtained in these ways is folded under controlled conditions in buffers containing reduced and oxidized glutathione. The resulting mixture may contain isomers with different pairings; the correct isomer is separated by chromatography and verified by checking whether it elutes at the same point when run together with the natural peptide. The potassium channel peptide family and its naming scheme were introduced in the article on α-KTx.
