Anemones punch holes: Toxins by NMR

Skip to Navigation


  • Published: Apr 1, 2017
  • Author: David Bradley
  • Channels: NMR Knowledge Base
thumbnail image: Anemones punch holes: Toxins by NMR

Protein with punch

Rockpool photo by David Bradley. Thumbnail credit: Separovic et al Molecules. 2017, 22(4). pii: E559. doi: 10.3390/molecules22040559

A new approach to constructing a sea anemone toxin makes it plausible to incorporate site-specific isotopic labels for solid-state nuclear magnetic resonance (NMR) spectroscopic structural studies that will ultimately reveal how it functions and provide clues as to the action of other pore-forming proteins.

Creatures that come into contact with the stinging tentacles of the sea anemone Actinia equine are liable to be stung as the beadlet anemone injects its venom to keep predators and those that invades its rock pool space at bay. The toxin produced by this marine creature and essentially injected sets about punching holes in your cell membranes at the molecular level. The toxin in question, equinatoxin II (EqtII) belongs to a unique family of 20-kilodalton toxins that form "pores" in cell membranes by preferentially binding to membranes containing sphingomyelin. These pores are cation-selective transporters of substances in and out of the cell membrane causing serious damage at the molecular level.

Previously, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy had revealed the structures of equinatoxin II, an actinoporin, isolated from A equine. Subsequent work then provided evidence for how this toxin punches holes in cell membranes causing inflammation and pain as the creature attempts by chemical proxy to ward off predators and unwitting paddlers who might return and cause it harm. Researchers from the UK, Spain, and Slovenia, and elsewhere unravelled the mode of action of this pore-forming protein which consists of a beta-sheet core with a flexible alpha-helical N-terminal domain.

Microwaved synthesis

Now, chemists John Karas, Marc-Antoine Sani, and Frances Separovic of the University of Melbourne, Melbourne, Victoria, Australia, have synthesised an 85 residue analogue of EqtII using a microwave-assisted technique. They did so by first assembling peptide precursors with over 40 residues using solid phase synthesis and then modifying the fragment to incorporate appropriate functionality and then potentially hook the pieces together. The researchers used circular dichroism (CD) spectroscopy to look at the N-terminal domain of EqtII(1–46) and EqtII(1–85) and showed that it keeps its predominantly alpha-helical structure in solution and also in the presence of lipid micelles. "This demonstrates the feasibility of assembling the full 179 residue protein EqtII via chemical means, the team says. They add that it is possible to add site-specific isotopic labels that can be exploited in subsequent solid-state NMR spectroscopic studies. Before that, the team must now optimise all their ligation and desulfurization reactions.

Toxic mechanism

The interaction of such proteins with cell membranes has been the subject of intense study for many years given that they might provide insights not only into toxicity mechanisms of the venoms of marine creatures and other animals but in looking for parallels with human membrane proteins many of which cannot be easily crystallised and so cannot be studied with crystallographic techniques. The new work provides insights into the fundamental molecular biology of an intriguing and apparently unique pore-forming toxin but given that the pore it forms is not yet well characterised the synthesis of such an analogue is an important goal as it might allow models to be built that get to the heart of the pore. Such work will then increase our overall understanding of membrane biochemistry and perhaps hint at how natural, non-toxin pore proteins of the kind that are impossible or difficult to crystallise and so examine with crystallography might also function. Given the importance of membrane proteins, pore-forming proteins, both endogenous and of the kind synthesised by pathogens and sea creatures, there are numerous steps to be taken in this area. Although if you're taking those steps in warm, shallow seas with lots of marine life, wear beach shoes and tread carefully.

Related Links

Molecules 2017, 22, 559: "Chemical Synthesis and Characterization of an Equinatoxin II(1-85) Analogue"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

Follow us on Twitter!

Social Links

Share This Links

Bookmark and Share


Suppliers Selection
Societies Selection

Banner Ad

Click here to see
all job opportunities

Copyright Information

Interested in separation science? Visit our sister site

Copyright © 2019 John Wiley & Sons, Inc. All Rights Reserved