The Role of Acidic Residues and the Proton Motive Force in Membrane Protein Assembly

细菌质膜由脂质双分子层和相关蛋白组成，是细菌细胞的主要渗透性屏障，将细胞质与细菌的环境分开。在细菌细胞的生命周期中，新的蛋白质必须部分插入到膜中（在膜蛋白的情况下）或完全插入（在跨膜易位的分泌蛋白的情况下）。人们认识到，不同的蛋白质利用不同的机制来实现这种插入或易位，并且已经描述了各种蛋白质易位机制。达尔贝博士实验室的总体目标是了解蛋白质如何插入或穿过膜，并获得正确的不对称拓扑结构。细菌的质膜实际上是一个带电的（通电的）电容器，具有电势和氢离子（质子）浓度梯度。储存在这个电容器中的能量被称为质子动力，或PMF，并作为在膜上发生的各种细菌代谢活动的能量来源。该项目解决了PMF在将新合成的蛋白质插入细菌膜中的作用。目前人们对这一作用知之甚少。PMF可能发挥多种作用：它可能直接影响膜蛋白的易位；它可以激活蛋白质机制促进插入；或者两者的某种组合。最近，研究表明细菌中带负电荷的氨基酸（“残基”）跨膜易位可以由PMF驱动，蛋白质或肽内带负电荷的残基可以在易位过程中发挥积极作用。这些结果为类似电泳的膜转移机制提供了证据。然而，目前尚不清楚所观察到的PMF的要求是特定于电成分（穿过膜的外部正电荷）还是跨膜pH成分（外部酸性，由外部浓度较高的氢离子浓度梯度引起）。本提案的目的是：确定PMF是否可以直接作用于膜蛋白底物，促进膜蛋白自发插入脂质体；确定膜蛋白的插入是否需要一种可能介导PMF效应的蛋白质成分；检查负电荷残基在蛋白质易位中的一般重要性；并确定PMF的哪些成分驱动带负电荷残基的易位。遗传学、生物化学和生物物理方法的结合将用于实现这些目标。这些研究很重要，因为它们将有助于确定膜蛋白的亲水区域在生物膜的脂质双分子层上移动的基本机制。这将扩展我们对基本细胞功能的知识和理解，这反过来将对生物技术的一般基础，特别是关于基因工程蛋白的生物加工作出重大贡献。

The bacterial plasma membrane, consisting of a lipid bilayer with associated proteins, is the prime permeability barrier for the bacterial cell, separating the cytoplasm from the bacterium's environment. During the life of the bacterial cell, new proteins must be inserted into the membrane, either partially (in the case of membrane proteins) or completely (in the case of secretory proteins that are translocated across the membrane). It is recognized that different proteins utilize different mechanisms to achieve this insertion or translocation, and a variety of protein translocation mechanisms have been described. The overall goal of Dr. Dalbey's laboratory is to understand how proteins insert into or across membranes and achieve their correct asymmetric topologies. The bacterial plasma membrane is in fact a charged (energized) capacitor, with both an electrical potential and a hydrogen ion (proton) concentration gradient across it. The energy stored in this capacitor is termed the proton motive force, or PMF, and serves as the energy source for a variety of bacterial metabolic activities that take place at the membrane. This project addresses the role of the PMF in the insertion of newly synthesized proteins into bacterial membranes. This role is presently poorly understood. There are a number of possible roles the PMF may play: it may directly affect the translocating membrane protein; it may activate a protein machinery to promote insertion; or some combination of the two. Recently, it has been shown in bacteria that the translocation of negatively charged amino acids ("residues") across the membrane can be driven by the PMF, and that negatively charged residues within a protein or peptide can play an active role in the translocation process. These results provide evidence for an electrophoresis-like membrane transfer mechanism. However, it is still not known whether the observed requirement for a PMF is specific for the electrical component (externally positive electrical charge across the membrane) or the transmembrane pH component (externally acidic, resulting from the hydrogen ion concentration gradient where the concentration is higher on the outside). The aims of this proposal are to: determine whether the PMF can act directly on the membrane protein substrate to promote the spontaneous insertion of membrane proteins into liposomes; determine whether the insertion of membrane proteins requires a protein component that may mediate the PMF effects; examine the general importance of negatively charged residues in protein translocation; and determine which components of the PMF drives translocation of negatively charged residues. A combination of genetic, biochemical and biophysical methods will be used to achieve these aims. These studies are important because they will help define a basic mechanism by which hydrophilic regions of membrane proteins move across the lipid bilayer of biological membranes. This will expand our knowledge and understanding of a basic cellular function, which in turn will contribute significantly to the general foundations of biotechnology, particularly with regard to bioprocessing of genetically engineered proteins.