Walker B, Bayley H
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545, USA.
Protein Eng. 1995 May;8(5):491-5. doi: 10.1093/protein/8.5.491.
In previous studies, the replacement of His35 in the pore-forming protein alpha-hemolysin (alpha HL) with Leu, Ile, Pro, Arg, Ser, Thr or Cys yielded inactive polypeptides. Here, we show that modification of the inactive single-cysteine mutant alpha HL-H35C with iodoacetamide, to form H35CamC, generates significant pore-forming activity. The closely related polypeptides H35N and H35Q have, respectively, essentially no activity and greatly reduced activity. The modified residue in H35CamC, S-carboxamidomethyl-cysteine, mimicks histidine in volume, polarity and hydrogen bonding potential, but is unable to ionize. Unmodified H35C is defective in the final step of pore formation: the conversion of an inactive heptameric membrane-bound assembly intermediate to a structure containing open channels. It is this step in assembly that is ameliorated in H35CamC.
在先前的研究中，将成孔蛋白α-溶血素（αHL）中的His35替换为Leu、Ile、Pro、Arg、Ser、Thr或Cys会产生无活性的多肽。在此，我们表明用碘乙酰胺修饰无活性的单半胱氨酸突变体αHL-H35C，形成H35CamC，会产生显著的成孔活性。密切相关的多肽H35N和H35Q分别基本没有活性和活性大大降低。H35CamC中修饰的残基S-羧酰胺甲基半胱氨酸在体积、极性和氢键潜力方面模拟组氨酸，但不能电离。未修饰的H35C在孔形成的最后一步存在缺陷：将无活性的七聚体膜结合组装中间体转化为含有开放通道的结构。正是组装中的这一步在H35CamC中得到了改善。
