Membrane Biophysical Studies of the Colicin E1 Channel

大肠杆菌素是由某些大肠杆菌菌株产生的有毒蛋白质。它们通常是在细菌细胞生长过程中对许多代谢挑战做出反应而产生的，包括DNA损伤、厌氧、分解代谢物抑制和营养耗尽。大肠杆菌素E1（colE 1）是通道形成大肠杆菌素的成员，其还包括大肠杆菌素A、B、Ia、Ib、N和K。大肠杆菌素的三维结构揭示了三个结构域的存在：受体结合、易位和通道结构域。受体结合域通过与细菌外膜上的维生素B12受体结合来启动毒素进入靶细胞。接下来，易位结构域与三聚体β-桶TolC外膜受体缔合，大肠杆菌素通过所述外膜受体转运到靶细菌的周质中。在一系列步骤中，colE 1依次展开、结合并自发插入膜中，形成封闭的通道状态。该通道在跨膜负电位的存在下打开，并通过Na+、K+和H+等细胞离子的逃逸引起宿主细胞质膜的去极化。目的：我们NSERC资助的研究项目的总体目标是：（1）确定colE 1通道蛋白的膜结合拓扑结构（2）建立新的荧光分析方法，用于colE 1膜蛋白的分析（低分辨率）结构，包括FRET三角法（3）为了揭示膜电位对colE 1离子通道的结构和功能的影响的分子细节，意义：所提出的研究将提供对colE 1沿着与其它相关通道形成大肠杆菌素（A、B、E1、Ia、Ib、N等）的通道状态的分子洞察。由于核磁共振和X射线研究未能产生高分辨率的膜结合态的结构，有必要采用其他方法来提供“低分辨率”的数据，但重要的信息，为了解大肠杆菌素的功能。此外，我们很可能会产生一个通用的方法来研究膜电位的结构和功能的整体和外周膜蛋白。

Colicins are toxic proteins produced by certain strains of E. coli during bacterial cell growth and they are often produced in response to a host of metabolic challenges, including DNA damage, anaerobiosis, catabolite repression and nutrient depletion. Colicin E1 (colE1) is a member of the channel-forming colicins, which also includes colicins A, B, Ia, Ib, N and K. The three-dimensional structures of colicins reveal the presence of three domains: receptor binding, translocation, and channel domains. The receptor-binding domain initiates toxin entry into target cells by binding with the vitamin B12 receptor on the bacterial outer membrane. Next, the translocation domain associates with the trimeric beta-barrel TolC outer membrane receptor, through which the colicin is transported into the periplasm of the target bacterium. In a series of steps, colE1 sequentially unfolds, binds and spontaneously inserts into the membrane to form a closed channel state. The channel opens in the presence of a trans-negative membrane potential and causes depolarization of the host cell cytoplasmic membrane through the escape of cellular ions such as Na+, K+, and H+.Objectives: The overall goals of our NSERC-funded research program are:(1) To determine the membrane-bound topology of the colE1 channel protein (closed channel state),(2) To develop new fluorescence methods for the elucidation of colE1 membrane protein (low-resolution) structure, including the FRET Triangulation Method(3) To reveal the molecular details of the effect of a membrane potential on the structure and function of the colE1 ion channel with application to membrane proteins, in general.Significance: The proposed studies will provide molecular insight into the channel states of colE1 along with other related channel-forming colicins (A, B, E1, Ia, Ib, N, etc). Since NMR and X-ray studies have failed to produce high-resolution structures of the membrane-bound states, it is necessary to employ other approaches to provide "low-resolution" data, but important information for the understanding of colicin function. Furthermore, it is likely that we will produce a general method for the study of membrane potential on the structure and function of both integral and peripheral membrane proteins.