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

细菌种类的多样性是巨大的，而存在于其膜中的脂质的多样性和分布突出了这种多样性。脂类是疏水化学物质，可以在链长、饱和度、分支和头基电荷等方面发生变化；所有这些都会导致膜厚度、净电荷和流动性的变化。脂质变化也可以作为一种分类和区分细菌种类的方法。尽管有这些知识，我们对脂质如何影响细菌膜功能的理解仍然知之甚少。******各种各样的膜蛋白必须在不同的脂质膜内发挥作用，以执行细胞的基本功能。膜蛋白如转运蛋白促进物质穿过这些脂质屏障的运动。转运蛋白如何在由不同脂质组成的膜中折叠和起作用尚不清楚。******我的研究计划的长期目标是研究细菌脂质组成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芽孢杆菌膜中发现的多配体转运体的功能上。构成各物种膜的脂质在脂肪酸类型、双层厚度和净电荷方面有显著差异。每个物种膜的独特差异是比较转运蛋白功能和折叠在不同的脂质组成中，通过监测底物运输的损失和收益，改变整体细胞生长的理想选择。***目的1：比较大肠杆菌和枯草芽孢杆菌在不同温度、pH和盐度下的天然脂质变化；已知改变脂质含量并可能调节转运蛋白折叠和功能的条件。***目的2：在每个物种中建立一个表达系统，通过异种共同表达特定脂质生物合成和转运基因，故意改变特定脂质和转运蛋白的含量。脂质变化的后果将通过观察细菌膜中转运蛋白折叠和拓扑结构的变化来监测。***目的3：利用转运蛋白序列数据对细菌脂质组成进行生物信息学和统计学比较，以确定脂质组成如何影响面向脂质区域的氨基酸保存和进化。******这个项目的意义和影响将提供关于脂质如何潜在地调节膜蛋白活性以及生长条件如何影响脂质含量的见解。了解膜蛋白如何在不同的脂质环境中折叠和功能，不仅进一步加深了我们对这些蛋白质如何工作的基本知识，而且为工业和学术应用确定了更好的生物工程和分离膜蛋白的方法。**

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. **