Abstract
The chemical synthesis of an 85 residue analogue of the pore-forming protein, Equinatoxin II (EqtII), was achieved. Peptide precursors with over 40 residues were assembled by solid phase synthesis. The EqtII(1–46) fragment was modified to the reactive C-terminal thioester and native chemical ligation was performed with the A47C mutated EqtII(47–85) peptide to form the EqtII(1–85) analogue. Circular dichroism spectroscopy indicated that the N-terminal domain of EqtII(1–46) and EqtII(1–85) maintains predominantly an α-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. Site-specific isotopic labels could be incorporated for structural studies in membranes by solid-state NMR spectroscopy.
Highlights
Equinatoxin II (EqtII), from the sea anemone Actinia equina is a 179 residue pore-forming toxin (PFT) and member of the actinoporin family [1,2]
EqtII fragments 1-46 and 47-85 were isolated as C-terminal hydrazides with the latter possessing an A47C mutation. 3-Methylpentyl protection was used for the βcarboxylate of aspartate residues instead of tert.-butyl to minimize aspartimide formation and its associated side products [26]
The reaction was monitored by reversed-phase high performance liquid chromatography (RP-HPLC) (Figure 3(a)) and the product was characterized via analytical RPHPLC and electro-spray ionization mass spectrometry (Figures 3(b) and 3(c), respectively)
Summary
Equinatoxin II (EqtII), from the sea anemone Actinia equina is a 179 residue pore-forming toxin (PFT) and member of the actinoporin family [1,2]. Solid-state NMR spectroscopy is a powerful tool for observing the molecular dynamics of proteins in membranes [8,9] This technique is often enhanced by incorporating “NMRactive” nuclei such as carbon-13 and nitrogen-15 into a biologically expressed product [4], which significantly enhances the signal and simplifies interpretation of the spectra. Our long-term goal is to chemically synthesize labelled carbon-13 and nitrogen-15 labelled EqtII analogues for 2D NMR experiments in model membranes [11,12] This will allow for the incorporation of isotopically enriched amino acids at peripheral residues to detect intermolecular contacts between monomers. Using this strategy would make it feasible to produce EqtII from 4 fragments that range from 39 to 52 residues (Figure 1)
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