Export of Protein in Escherichia coli

描述（由申请人提供）：特定多肽跨膜易位对所有活细胞都是必不可少的。从单细胞生物到哺乳动物，大肠杆菌中异源三聚体复合物SecYEG提供的膜转移途径在所有三个生命领域都是高度保守的。除了在几乎所有情况下通过膜的途径外，无论该过程发生在原核生物还是真核生物中，伴侣蛋白都参与了要运输的前体多肽结合的初始阶段。多肽通过膜的捕获和运动现象的基本原理可能在不同的系统中是共享的。因此，我们通过研究细菌出口所获得的知识将为其他出口系统的分子机制提供见解。该研究计划旨在阐明该过程的机制细节，重点是前体多肽沿着从伴侣SecB到SecA的途径，系统的atp酶马达以及通过SecYEG易位的动力学。这项工作将增加对分子开关的理解深度，包括复合物内特定接触的变化，这对前体从一个结合伙伴移动到另一个结合伙伴至关重要，以及SecA内的构象变化，这是结合能量和ATP水解转化为通过膜通道易位的机械功的基础。该研究策略利用生物物理和生物化学技术的平衡，相互补充。实验的范围从用滴定量热法测定结合参数，用光散射和超离心测定配合物的流体动力学性质，到将整个易位系统完全重构为蛋白质脂质体，该系统不仅包括易位，还包括辅助蛋白SecDF/YajC以及作为产生原动力手段的细菌视紫红质。其中的创新技术包括纳米圆盘的使用，它允许像SecYEG这样的膜蛋白被当作可溶性颗粒来处理，以及单分子原子力显微镜的使用。许多令人感兴趣的问题不能用集合方法轻易地回答，因为解释被沿着出口路径发生的多重动态平衡所混淆。对于这些研究来说，用单分子技术评估复合物是必不可少的。该研究计划的一个总体优势是，所有方法都是在尽可能接近生理的溶液条件和蛋白质浓度下进行的。这个建议是基于我们过去的研究，应该使该领域更接近于蛋白质定位的分子描述。

DESCRIPTION (provided by applicant): Translocation of specific polypeptides across membranes is essential for all living cells. The pathway of transfer through the membrane provided by the heterotrimeric complex SecYEG in Escherichia coli is highly conserved in all three kingdoms of life, from single cell organisms to mammals. In addition to a pathway through the membrane in almost all cases, whether the process occurs in prokaryotes or eukaryotes, chaperones are involved in the initial stages of binding of precursor polypeptides that are to be transported. The principles underlying the phenomena of capture and movement of polypeptides through membranes are likely to be shared among different systems. Therefore what we learn by studying bacterial export will provide insights into the molecular mechanisms of other export systems. The research plan is designed to elucidate the mechanistic details of the process with emphasis on the dynamics of passage of a precursor polypeptide along the pathway from the chaperone SecB to SecA, the ATPase motor of the system, and on through the SecYEG translocon. The work will increase the depth of understanding of molecular switches including both changes in specific contacts within complexes that are crucial to the movement of the precursor from one binding partner to another as well as conformation changes within SecA that underlie the conversion of energy of binding and hydrolysis of ATP to the mechanical work of translocation through the channel in the membrane. The research strategy makes use of a balance of biophysical and biochemical techniques that complement one another. The experiments proposed range from determinations of binding parameters by titration calorimetry and of hydrodynamic properties of complexes by light scattering and ultracentrifugation to the complete reconstitution into proteoliposomes of the entire translocation system comprising not only the translocon, but also the accessory proteins SecDF/YajC as well as bacterial rhodopsin as a means to generate protonmotive force. Among the innovative techniques are the use of Nanodiscs that allow membrane proteins such as SecYEG to be treated as a soluble particle and the use of single molecule atomic force microscopy. Many questions of interest cannot be readily answered by ensemble methods because interpretation is confounded by the multiple dynamic equilibria that occur along the export pathway. For these studies assessment of complexes by a single molecule technique is essential. An overall advantage of the research plan is that all methods are carried out in solution conditions and protein concentrations that are as close as possible to physiological. The proposal is firmly based on our past research and should move the field nearer to a molecular description of protein localization. PUBLIC HEALTH RELEVANCE: Localization and folding of proteins is an essential process in all living cells and errors in the processes are the basis of many human diseases. The mechanism is highly conserved and thus what we learn by studying export in Escherichia coli will be applicable to the phenomenon in all cells from bacteria to humans.