Unnatural amino acid mutagenesis to probe ion channel structure and function

描述（由申请人提供）：拟议研究的长期目标是了解K+通道中的门控机制。K+通道对于可兴奋细胞产生电脉冲至关重要。它们一直是深入研究的主题，然而，仍然存在一些关键问题。这些问题仍然悬而未决的原因之一是传统的定点诱变以精确的方式修饰通道结构的能力有限。该方法允许限于二十种天然存在的氨基酸的组的氨基酸取代。通过非天然氨基酸诱变可以实现蛋白质的结构和电子性质的修饰的更高准确度。化学合成是非天然氨基酸诱变的有力方法，其提供了几乎无限的氨基酸侧链和肽骨架修饰的选择。使用传统的定点诱变，这样的修饰是不可能的。本申请的目的是发展化学合成离子通道蛋白的一般策略，并将其应用于钾通道失活过程的研究。在我的博士后研究中，我提议研究细菌K+通道KcsA中被称为“缓慢失活”的门控过程。我将追求两个主要的具体目标：目标1）优化化学合成方法，研究膜蛋白在体外。在我的博士后培训中开发的化学合成方案不仅对研究KcsA通道有用，而且还适用于其他重要类别的膜蛋白的化学合成。目的2）探讨化学合成KcsA的失活机制。我建议，一个更好的理解KcsA失活可以通过使用非天然氨基酸突变来实现。在我的研究中，我将使用化学合成来精确地干扰KcsA通道的选择性过滤器及其与周围蛋白质的相互作用。这种方法将允许我评估通道修饰对慢失活的影响，这将通过电生理测量来确定。我的研究将有助于建立缓慢失活过程的分子基础。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to understand gating mechanisms in K+ channels. K+ channels are critical to the generation of electrical impulses by excitable cells. They have been the subject of intense research, however, a number of key questions remain. One of the reasons why these questions are still open is the limited ability of traditional site-directed mutagenesis to modify the channel structure in a precise way. This approach allows amino acid substitutions that are limited to the set of twenty naturally occurring amino acids. A higher accuracy for modifications of the structural and electronic properties of the protein can be achieved by means of unnatural amino acid mutagenesis. Chemical synthesis is a powerful approach for unnatural amino acid mutagenesis that offers an almost unlimited choice of amino acid side chain and peptide backbone modifications. Such modifications are not possible using traditional site-directed mutagenesis. The goal of this application is to develop general strategies for the chemical synthesis of ion channel proteins and to apply it to investigation on the inactivation process in potassium channels. In my postdoctoral research, 1 proposed to investigate the gating process referred to as "slow inactivation" in the bacterial K+ channel, KcsA. I will pursue two major specific aims: Aim 1) To optimize chemical synthesis methodology for investigating membrane proteins in vitro. The chemical synthesis protocols that will be developed within my postdoctoral training will be useful not only for investigating the KcsA channel but will also find applicability in the chemical synthesis of other important classes of membrane proteins. Aim 2) To investigate the mechanism of KcsA inactivation using chemical synthesis. I propose that a better understanding of the KcsA inactivation may be achieved by using unnatural amino acid mutagenesis. For my research, I will use the chemical synthesis to precisely perturb the selectivity filter of the KcsA channel and its interactions with the surrounding protein. This approach will allow me to evaluate the effect of channel modifications on slow inactivation that will be determined by electrophysiological measurements. My research will help to establish the molecular basis for the process of slow inactivation.