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

细菌质膜由脂质双层和相关蛋白质组成，是细菌细胞的主要通透性屏障，将细胞质与细菌环境分开。在细菌细胞的生命过程中，必须将新的蛋白质部分(对于膜蛋白)或完全(对于跨膜转移的分泌蛋白)插入到膜中。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转位，并且已经描述了各种蛋白质转位机制。达尔贝博士实验室的总体目标是了解蛋白质如何插入或跨膜，并实现正确的不对称拓扑结构。细菌质膜实际上是一个带电的电容器，其上既有电势又有氢离子(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，作为发生在膜上的各种细菌新陈代谢活动的能量来源。该项目介绍了PMF在将新合成的蛋白质插入细菌膜中的作用。目前，人们对这一角色知之甚少。PMF可能扮演许多可能的角色：它可能直接影响转运膜蛋白；它可能激活促进插入的蛋白质机制；或者两者的某种组合。最近，在细菌中发现，带负电荷的氨基酸(“残基”)的跨膜转运可以由PMF驱动，蛋白质或多肽中的带负电荷的残基可以在转运过程中发挥积极作用。这些结果为电泳膜转移机制提供了证据。然而，尚不清楚观察到的PMF要求是电气成分(膜上的外部正电荷)还是跨膜pH成分(外部酸性，由氢离子浓度梯度造成，其中外部浓度较高)。这项建议的目的是：确定PMF是否能直接作用于膜蛋白底物以促进膜蛋白自发插入脂质体；确定膜蛋白的插入是否需要可能介导PMF效应的蛋白质组分；检查带负电荷残基在蛋白质转运中的一般重要性；以及确定PMF的哪些组分驱动带负电荷残基的移位。将结合遗传、生化和生物物理方法来实现这些目标。这些研究很重要，因为它们将有助于确定膜蛋白的亲水性区域通过生物膜脂双层移动的基本机制。这将扩大我们对基本细胞功能的知识和理解，这反过来将大大有助于生物技术的一般基础，特别是在基因工程蛋白质的生物处理方面。

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.