Chemical Synthesis and Characterization of an Equinatoxin II(1-85) Analogue

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.

Keywords: membrane protein structure; native chemical ligation; selectively labelled proteins; solid-phase peptide synthesis; solid-state NMR.

Figure 1

(a) Amino acid sequence of Equinatoxin II (EqtII). The alanine residues to be incorporated as cysteine are in bold; (b) Proposed strategy for the chemical assembly of native EqtII. Note that the scope of the work reported here involves the native chemical ligation (NCL) of the EqtII(1–46) and EqtII(47–85) to give the EqtII(1–85) analogue. The crystal structure of full-length EqtII (PDB: 1IAZ) is shown on the right.

Figure 2

Reaction scheme for the formation of the EqtII(1–85) analogue. (a): pH 7, 20 °C, 18 h; (b): reversed-phase high performance liquid chromatography (RP-HPLC).

Figure 3

(a) NCL reaction after 4 h; (b) analytical RP-HPLC of the purified EqtII(1–85) analogue and (c) ESI-MS of the purified EqtII(1–85) analogue (inset: Deconvoluted spectrum; molecular mass (calc.) = 9091.4 Da). Please note that chromatograms (a,b) were analyzed using different HPLC instruments, which accounts for the small difference in the retention time of the EqtII(1–85) analogue.

Figure 4

Circular dichroism (CD) spectroscopy of the: (a) EqtII(1–46) fragment; (b) EqtII(47–85) A47C fragment; (c) EqtII(1–85) fragment (as the 4-mercaptophenylacetic acid (MPAA) adduct). ~40 μM peptide/protein in buffer, 15 mM dodecylphosphocholine (DPC) or sodium dodecyl sulfate (SDS) micelles, pH 7.2, 25 °C.