National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.
BMB Rep. 2013 Mar;46(3):175-80. doi: 10.5483/bmbrep.2013.46.3.100.
Cyt2Aa2 is a mosquito larvicidal and cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. The toxin becomes inactive when isoleucine at position 150 was replaced by alanine. To investigate the functional role of this position, Ile150 was substituted with Leu, Phe, Glu and Lys. All mutant proteins were produced at high level, solubilized in carbonate buffer and yielded protease activated product similar to those of the wild type. Intrinsic fluorescence spectra analysis suggested that these mutants retain similar folding to the wild type. However, mosquito larvicidal and hemolytic activities dramatically decreased for the I150K and were completely abolished for I150A and I150F mutants. Membrane binding and oligomerization assays demonstrated that only I150E and I150L could bind and form oligomers on lipid membrane similar to that of the wild type. Our results suggest that amino acid at position 150 plays an important role during membrane binding and oligomerization of Cyt2Aa2 toxin.
Cyt2Aa2 是由苏云金芽孢杆菌亚种。darmstadiensis 产生的一种杀蚊幼虫和细胞溶解毒素。当 150 位的异亮氨酸被丙氨酸取代时，毒素失去活性。为了研究该位置的功能作用，将 Ile150 替换为 Leu、Phe、Glu 和 Lys。所有突变蛋白均高水平表达，可溶于碳酸盐缓冲液中，并产生与野生型相似的蛋白酶激活产物。内源荧光光谱分析表明，这些突变体保留了与野生型相似的折叠。然而，I150K 的杀蚊幼虫和溶血活性显著降低，而 I150A 和 I150F 突变体则完全丧失活性。膜结合和寡聚化实验表明，只有 I150E 和 I150L 能够结合并在脂质膜上形成类似于野生型的寡聚体。我们的结果表明，150 位的氨基酸在 Cyt2Aa2 毒素的膜结合和寡聚化过程中起着重要作用。
