Polyhydroxybutyrate/Polyphosphate Complexes as Conductive Cores of Ion Channels

聚羟基丁酸酯/聚磷酸盐复合物作为离子通道的导电核心

• 批准号: 0422938
• 负责人: Rosetta Reusch
• 金额: --
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0422938&HistoricalAwards=false
• 关键词: Polyhydroxybutyrate; Polyphosphate; Complexes; Conductive; Cores

Intellectual merit. The premise of this research is that complexes of two ubiquitous polymers, 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 selective ion channels in planar lipid bilayers. This project will examine the putative modification of inherent 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. Consequently, cPHB/polyP complexes are highly selective for divalent cations above pH 7.0, but become selective for monovalent cations when the pH is below 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 project 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. In terms of training and education, postdoctoral, graduate and undergraduate students from Michigan State University and other institutions will be involved in various aspects of 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.

智力上的优点。 本研究的前提是两种普遍存在的聚合物聚（R）-3-羟基丁酸酯和聚磷酸盐（cPHB/polyP）的复合物形成蛋白质离子通道的导电核心。先前已经表明，从细菌质膜分离或从合成聚合物制备的cPHB/聚P复合物在平面脂质双层中产生选择性离子通道。本项目将研究变铅青链霉菌钾通道KcsA多肽对固有cPHB/polyP通道选择性的假定修饰。 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/聚P复合物在pH 7.0以上对二价阳离子具有高度选择性，但在pH低于7.0时对一价阳离子具有选择性。 KcsA在室温下在平面双层中表现出类似的阳离子选择性;通道在pH 7.2下优选二价阳离子，但在pH 6.8下对K+具有高度选择性。 然而，在高于26 ℃的温度下，KcsA对K+具有高度选择性，而与pH无关。假设KcsA经历热转变，导致碱性残基位于KcsA多肽的细胞内链的末端，靠近聚P的末端单位;这些残基上的正电荷对聚P的选择性的影响将与降低的pH相当。在该项目中，通过PCR定点突变制备这些碱性残基的突变体，蛋白质将在E. coli中进行亲和层析纯化。 然后将平面脂质双层中这些突变KcsA通道的阳离子选择性与野生型KcsA的阳离子选择性进行比较以检验假设。 更广泛的影响。 该项目提供了一种新的离子传输机制。 此外，cPHB/polyP通道是DNA转运的假定载体，它们可以作为阳离子、pH或温度的纳米传感器。在培训和教育方面，来自密歇根州立大学和其他机构的博士后、研究生和本科生将参与本项研究的各个方面。此外，还将开发一个网站，提供有关cPHB和polyP及其复合物的初级和高级信息，以促进学生，科学家和公众更好地了解这些基本细胞成分的重要结构和代谢作用。