Export of Protein in Escherichia coli

描述（由申请人提供）：特异性多肽跨膜转运对所有活细胞都是必需的。通过异源三聚体复合物SecYEG在大肠杆菌中提供的膜转移途径在所有三个生命界中是高度保守的，从单细胞生物到哺乳动物。除了在几乎所有情况下通过膜的途径之外，无论该过程发生在原核生物还是真核生物中，伴侣蛋白都参与待转运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共享。因此，我们通过研究细菌输出所学到的知识将为其他输出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分子伴侣SecB到SecA的途径（系统的ATP酶马达）以及通过SecYEG易位子的动力学。这项工作将增加对分子开关的理解的深度，包括复合物内特定接触的变化，这些变化对前体从一个结合伴侣到另一个结合伴侣的运动至关重要，以及SecA内的构象变化，这些变化是ATP结合和水解能量转换为通过膜中通道易位的机械功的基础。 研究战略利用了生物物理和生物化学技术的平衡，相互补充。建议的实验范围从滴定量热法结合参数的测定和配合物的流体动力学性质的光散射和ultracentraggation到完整的重组成整个易位系统的蛋白脂质体，不仅包括translocon，但也辅助蛋白SecDF/YajC以及细菌视紫红质作为一种手段，以产生质子动力。其中的创新技术是使用纳米盘，使膜蛋白，如SecYEG被视为可溶性颗粒和使用单分子原子力显微镜。许多感兴趣的问题不能很容易地回答合奏方法，因为解释是混淆的多重动态平衡发生沿着出口途径。对于这些研究，通过单分子技术评估复合物是必不可少的。该研究计划的总体优势在于，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。 该建议是坚定地基于我们过去的研究，并应移动该领域更接近蛋白质定位的分子描述。 公共卫生相关性：蛋白质的定位和折叠是所有活细胞中必不可少的过程，并且该过程中的错误是许多人类疾病的基础。该机制是高度保守的，因此我们通过研究大肠杆菌中的输出所学到的知识将适用于从细菌到人类的所有细胞中的现象。

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.