Dynamics of the Lactose Permease of Escherichia coli

描述（由申请人提供）：离子梯度偶联主动转运是一种在健康和疾病中发挥重要作用的生物现象。多年来，该实验室一直专注于大肠杆菌的乳糖通透酶（LacY）作为这类转运蛋白的范例。为了更详细地理解传输机制，需要原子级的结构以及时间分辨的动态信息。最近，在3.5A左右解出了LacY的内向构象的X射线结构，LacY是包含超过3000个成员的主要易化剂超家族的成员。这种非常疏水的蛋白质由两个对称的6-螺旋束组成，具有巨大的亲水性内腔，其中包含糖结合位点并且仅对细胞质开放。与此同时，在DK 51131的部分资助下，Pi的实验室还构建并表征了LacY每个位置的单Cys突变体库，以及其他定点突变体，以及一系列广泛使用的定点技术，这些技术可以很容易地应用于动态研究。基于结构和大量的生物化学和生物物理学证据，已经提出了一种机制，其中含有结合位点的亲水性空腔可交替地进入膜的两侧。此外，质子电化学梯度已被证明通过加速限速步骤（质子的解离）而不是改变膜两侧结合位点的亲和力来驱动糖积累对抗浓度梯度。因此，所取得的进展使该实验室处于一个独特的位置，可以研究LacY催化的运输的详细机制。在这个建议中，我们将使用合理设计的突变体的基础上的X-射线结构的LacY结合各种生物化学和生物物理技术在手，以获得时间分辨的数据，将产生动态信息的构象状态的LacY和它们的相互转换在配体结合和营业额。

DESCRIPTION (provided by applicant): Ion-gradient coupled active transport is a biological phenomenon that plays important roles in health and disease. For a number of years, this laboratory has focused on the lactose permease of Escherichia coli (LacY) as a paradigm for this class of transport proteins. In order to understand the transport mechanism in greater detail, structure at the atomic level, as well as time-resolved dynamic information, is required. Recently, an x-ray structure of the inward-facing conformation of LacY, a member of the Major Facilitator Super-family containing over 3000 members, was solved at about 3.5 A. This very hydrophobic protein is composed of two symmetrical 6-helix bundles with a huge, hydrophilic internal cavity that contains the sugar binding site and is open to the cytoplasm only. At the same time, financed in part by DK 51131, the Pi's laboratory has also constructed and characterized a library of single-Cys mutants at each position of LacY, as well as other site-directed mutants, and a battery of widely used site-directed techniques that can be easily applied to dynamic studies. Based on the structure and a large body of biochemical and biophysical evidence, a mechanism has been proposed in which the hydrophilic cavity containing the binding site is alternatively accessible to either side of the membrane. Moreover, the proton electrochemical gradient has been shown to drive sugar accumulation against a concentration gradient by accelerating a rate-limiting step (dissociation of the proton) rather than changing the affinity of the binding site on either side of the membrane. Thus, the progress achieved places this laboratory in a unique position to study the detailed mechanism of transport catalyzed by LacY. In this proposal, we will use rationally designed mutants based on the x-ray structure of LacY in conjunction with various biochemical and biophysical techniques at hand to obtain time-resolved data that will yield dynamic information regarding the conformational states of LacY and their interconversion during ligand binding and turnover.