Komarov Alexander G, Costantino Corey A, Valiyaveetil Francis I
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR, USA.
Methods Mol Biol. 2013;995:3-17. doi: 10.1007/978-1-62703-345-9_1.
Potassium channels conduct K(+) ions selectively and at very high rates. Central to the function of K(+) channels is a structural unit called the selectivity filter. In the selectivity filter, a row of four K(+) binding sites are created using mainly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, site-directed mutagenesis is of limited utility in investigating the selectivity filter. In order to overcome this limitation, we have developed a semisynthetic approach, which permits the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that we have developed for the semisynthesis of the K(+) channel, KcsA. We anticipate that the protocols described in this chapter will also be applicable for the semisynthesis of other integral membrane proteins of interest.
钾通道能够以非常高的速率选择性地传导钾离子。钾通道功能的核心是一个称为选择性过滤器的结构单元。在选择性过滤器中，主要利用主链羰基氧原子形成一排四个钾离子结合位点。由于蛋白质主链的参与，定点诱变在研究选择性过滤器方面的作用有限。为了克服这一限制，我们开发了一种半合成方法，该方法允许使用化学合成来操纵选择性过滤器。在本章中，我们描述了我们为钾通道KcsA的半合成所开发的方案。我们预计本章所述的方案也将适用于其他感兴趣的整合膜蛋白的半合成。
