Karas John A, Sani Marc-Antoine, Separovic Frances
School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC 3010, Australia.
Molecules. 2017 Mar 30;22(4):559. doi: 10.3390/molecules22040559.
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.
成功实现了成孔蛋白海葵毒素II（EqtII）85个残基类似物的化学合成。通过固相合成组装了具有40多个残基的肽前体。将EqtII(1-46)片段修饰为反应性C末端硫酯，并与A47C突变的EqtII(47-85)肽进行天然化学连接，以形成EqtII(1-85)类似物。圆二色光谱表明，EqtII(1-46)和EqtII(1-85)的N末端结构域在溶液中以及在脂质微团存在的情况下主要保持α-螺旋结构。这证明了通过化学方法组装完整的179个残基蛋白EqtII的可行性。可以通过固态核磁共振光谱掺入位点特异性同位素标记用于膜结构研究。
