Membrane Biophysical Studies of the Colicin E1 Channel

Colicins是由某些菌株在细菌细胞生长过程中产生的有毒蛋白质，它们通常是在应对一系列代谢挑战时产生的，包括DNA损伤、厌氧、分解代谢抑制和营养耗竭。Colicin E1(Cole1)是形成通道的Colicins的一员，它还包括Colicins A、B、Ia、Ib、N和K。Colicin的三维结构揭示了Colicin的三个结构域：受体结合、易位和通道结构域。受体结合区通过与细菌外膜上的维生素B12受体结合，启动毒素进入靶细胞。接下来，易位结构域与三聚体β桶TolC外膜受体结合，通过该受体将结肠素运输到目标细菌的周质中。在一系列步骤中，cole1依次展开、结合并自发插入膜中，形成关闭的通道状态。该通道在膜电位为负的情况下开放，并通过逃逸细胞内的Na+、K+和H+等离子引起宿主细胞胞质膜的去极化。 目标：我们NSERC资助的研究计划的总体目标是： (1)确定colE1通道蛋白的膜结合拓扑(关闭通道状态)， (2)建立新的荧光方法研究cole1膜蛋白(低分辨率)结构，包括FRET三角法。 (3)通过对膜蛋白的应用，揭示膜电位对cole1离子通道结构和功能影响的分子细节。 意义：拟议的研究将提供对cole1以及其他相关的通道形成素(A、B、E1、Ia、Ib、N等)通道状态的分子洞察。由于核磁共振和X射线研究未能获得膜结合态的高分辨率结构，因此有必要使用其他方法来提供低分辨率数据，但这对于了解粘附素的功能是重要的信息。此外，我们有可能建立一种通用的方法来研究膜电位对整体膜蛋白和外周膜蛋白的结构和功能的影响。

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.