Komarov Alexander G, Linn Kellie M, Devereaux Jordan J, Valiyaveetil Francis I
Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health and Sciences University, Portland, Oregon, USA.
Methods Enzymol. 2009;462:135-50. doi: 10.1016/S0076-6879(09)62007-3.
The ability to selectively conduct K(+) ions is central to the function of K(+) channels. Selection for K(+) and rejection of Na(+) takes place in a conserved structural element referred to as the selectivity filter. The selectivity filter consists of four K(+)-specific ion binding sites that are created using predominantly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, experimental manipulation of the ion binding sites in the selectivity filter is not possible using traditional site directed mutagenesis. The limited suitability of the site-directed mutagenesis for studies on the selectivity filter has motivated the development of a semisynthesis approach, which enables the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that are presently used in our laboratory for the semisynthesis of the bacterial K(+) channel, KcsA. We show the introduction of a spectroscopic probe into the KcsA channel using semisynthesis. We also review previous applications of semisynthesis in investigations of K(+) channels. While the protocols described in this chapter are for the KcsA K(+) channel, we anticipate that similar protocols will also be applicable for the semisynthesis of other integral membrane proteins.
选择性传导钾离子的能力是钾离子通道功能的核心。对钾离子的选择和对钠离子的排斥发生在一个保守的结构元件中，称为选择性过滤器。选择性过滤器由四个主要利用主链羰基氧原子形成的钾离子特异性离子结合位点组成。由于蛋白质主链的参与，使用传统的定点诱变无法对选择性过滤器中的离子结合位点进行实验操作。定点诱变在选择性过滤器研究中的适用性有限，这促使了一种半合成方法的发展，该方法能够利用化学合成来操纵选择性过滤器。在本章中，我们描述了目前我们实验室用于细菌钾离子通道KcsA半合成的方案。我们展示了使用半合成将光谱探针引入KcsA通道的过程。我们还回顾了半合成在钾离子通道研究中的先前应用。虽然本章描述的方案是针对KcsA钾离子通道的，但我们预计类似的方案也将适用于其他整合膜蛋白的半合成。
