Polyhydroxybutyrate/Polyphosphate Complexes as Conductive Cores of Ion Channels

知识价值。本研究的前提是两种普遍存在的均聚物聚-(R)-3-羟基丁酸酯和聚磷酸盐（cPHB/polyP）的配合物形成蛋白质离子通道的导电核心。以前的研究表明，从细菌质膜分离或合成聚合物制备的cPHB/polyP复合物在平面脂质双层中产生阳离子选择性离子通道。本项目将检验lividans钾通道（KcsA）的多肽对cPHB/polyP通道选择性的固有修饰。磷酸盐在pH 7.0以上具有二价负电荷，在pH 7.0以下具有单价负电荷；因此，cPHB/polyP配合物对pH 7.0以上的二价阳离子具有高度选择性，在pH 7.0时不表现出偏好，而在pH小于7.0时对单价阳离子具有选择性。室温下，KcsA在平面双分子层中表现出相似的阳离子选择性；当pH值为7.2时，通道更倾向于二价阳离子，但当pH值为6.8时，通道对K+具有高度选择性。然而，在26℃以上的温度下，无论ph值如何，KcsA对K+都具有高度选择性。假设是KcsA经历了一个热转变，导致KcsA多肽的细胞内链末端的基本残基靠近polyP的末端单位；这些碱基上的正电荷对polyP选择性的影响将与降低ph的影响相当。在本项目中，这些碱基的突变体将通过PCR位点定向诱变制备；这些蛋白将在大肠杆菌中过表达，并通过亲和层析纯化。然后将这些突变KcsA通道在平面脂质双层中的阳离子选择性与野生型KcsA进行比较，以验证该假设。更广泛的影响。这一建议提供了一种新的离子传输机制。此外，cPHB/polyP通道被认为是DNA运输的载体，它们可以作为阳离子、pH或温度的纳米传感器。来自密歇根州立大学和其他机构的博士后、研究生和本科生参与了这项研究。此外，还将建立一个网站，提供有关cPHB和polyP及其复合物在初级和高级水平的信息，以促进学生、科学家和公众对这些基本细胞成分的重要结构和代谢作用有更深入的了解。

Intellectual merit. The premise of this research is that complexes of two ubiquitous homopolymers, poly-(R)-3-hydroxybutyrate and polyphosphate (cPHB/polyP), form the conductive cores of protein ion channels. It has previously been shown that cPHB/polyP complexes, isolated from bacterial plasma membranes or prepared from the synthetic polymers, create cation-selective ion channels in planar lipid bilayers. This project will examine the putative modification of intrinsic cPHB/polyP channel selectivity by polypeptides of the Streptomyces lividans potassium channel, KcsA. Phosphates have a divalent negative charge above pH 7.0 and a monovalent negative charge below pH 7.0; accordingly, cPHB/polyP complexes are highly selective for divalent cations above pH 7.0, display no preference at pH 7.0 and become selective for monovalent cations at pH less than 7.0. KcsA demonstrates similar cation selectivity in planar bilayers at room temperatures; the channels prefer divalent cations at pH 7.2, but are highly selective for K+ at pH 6.8. However, at temperatures above 26 C, KcsA is highly selective for K+, regardless of pH. The hypothesis is that KcsA undergoes a thermal transition that results in the placement of basic residues at the ends of the intracellular strands of KcsA polypeptides close to the end units of polyP; the effect of the positive charges on these residues on selectivity of polyP would be comparable to that of a lowered pH. In this project, mutants of these basic residues will be prepared by PCR site-directed mutagenesis; the proteins will be overexpressed in E. coli and purified by affinity chromatography. The cation selectivity of these mutant KcsA channels in planar lipid bilayers will then be compared to that of wild-type KcsA to test the hypothesis. Broader impact. This proposal offers a novel mechanism for ion transport. Moreover, the cPHB/polyP channels are putative vehicles for DNA transport and they may serve as nanosensors for cations, pH or temperature. Postdoctoral, graduate and undergraduate students from Michigan State University and other institutions are participants in this research. In addition, a web site will be developed to provide information about cPHB and polyP and their complexes at both elementary and advanced levels in order to foster a greater understanding of the important structural and metabolic roles of these fundamental cell constituents among students, scientists and the general public.