The influence of bacterial lipid composition variation on transporter function and folding.

细菌物种的多样性是巨大的，而驻留在其膜上的广泛种类和分布的脂类突出了这种多样性。脂类是一种疏水化学物质，可以改变链长、饱和度、支化和头基电荷；所有这些都会导致膜厚度、净电荷和流动性的变化。脂类的变化也可以用来分类和区分细菌种类。尽管有这些知识，我们对脂类如何影响细菌膜功能的了解仍然很少。各种膜蛋白必须在不同的脂膜中发挥作用，才能发挥细胞的基本功能。膜蛋白，如转运蛋白，促进物质跨越这些脂质屏障。转运蛋白如何在由不同脂质成分组成的膜中折叠和发挥作用还不是很清楚。*我的研究计划的长期目标是检测细菌脂类成分对转运蛋白功能和折叠的影响。为了实现这一目标，三个短期目标将集中在革兰氏阴性大肠杆菌和革兰氏阳性枯草杆菌膜中发现的多配体转运蛋白的功能上。组成每种膜的脂类在脂肪酸类型、双层厚度和净电荷方面存在显著差异。每种膜的独特差异是通过监测改变整体细胞生长的底物运输的损失和收益来比较不同脂质成分中的转运蛋白功能和折叠的理想选择。*目标1：比较在不同温度、pH和盐度下生长的大肠杆菌和枯草杆菌中的天然脂质变化；已知这些条件会改变脂质含量，并可能调节转运蛋白的折叠和功能。*目标2：在每个物种中建立一个表达系统，通过异源共表达特定的脂类生物合成和转运蛋白基因来故意改变特定的脂类和转运蛋白的含量。通过观察细菌膜上转运蛋白折叠和拓扑结构的变化，将在体内监测脂质变化的后果。*目标3：将细菌脂类组成与转运蛋白序列数据进行生物信息学和统计学比较，以确定脂组成如何影响面对脂类的区域的氨基酸保守和进化。*该计划的意义和影响将为脂类如何潜在地调节膜蛋白活性以及生长条件如何影响脂类含量提供见解。了解膜蛋白如何在不同的脂质环境中折叠和发挥功能，不仅加深了我们对这些蛋白如何工作的基础知识，而且确定了更好的方法来生物工程和分离膜蛋白，用于工业和学术应用。**

Bacterial species diversity is vast and the wide variety and distribution of lipids that reside in their membranes highlights this diversity. Lipids are hydrophobic chemicals that can vary in chain length, saturation, branching and head group charge; all of which result in changes in membrane thickness, net charge and fluidity. Lipid variation has can also be used as a way to classify and distinguish bacterial species from one another. Despite this knowledge, our understanding of how lipids influence the function of bacterial membranes remains poorly understood. ******A broad assortment of membrane proteins must function within diverse lipid membranes to perform essential functions for the cell. Membrane proteins such as transporters facilitate the movement of substances across these lipid barriers. How transporters fold and function in membranes composed of different lipid compositions is not well understood. ******The long-term objective of my research program is to examine the influence of bacterial lipid composition on transporter function and folding. To accomplish this, three short-term objectives will focus on the function of multi-ligand transporters found in the membranes of Gram-negative Escherichia coli and of Gram-positive Bacillus subtilis. Lipids that make up each species membrane differ significantly in fatty acid type, bilayer thickness and net charge. The unique differences of each species membrane are ideal to compare transporter function and folding in different lipid compositions by monitoring losses and gains in substrate transport that alter overall cell growth. ***Objective 1: Involves a comparison of natural lipid variation in E. coli and B. subtilis grown at different temperatures, pH and salinity; conditions which are known to alter lipid content and potentially regulate transporter folding and function. ***Objective 2: Develops an expression system in each species to deliberately vary the content of specific lipids and transporters by heterologous co-expression of particular lipid biosynthetic and transporter genes. The consequences of lipid variation will be monitored in vivo by observing changes in transporter folding and topology in bacterial membranes. ***Objective 3: Bioinformatic and statistical comparison of bacterial lipid compositions will be made with transporter protein sequence data to determine how lipid compositions influence amino acid conservation and evolution of regions that face lipids. ******The significance and impact of this program will provide insights into how lipids potentially regulate membrane protein activity and how growth conditions influence lipid content. Knowledge of how membrane proteins fold and function in diverse lipid environments not only furthers our fundamental knowledge of how these proteins work, but identifies better ways to bioengineer and isolate membrane proteins for industrial and academic applications. **