Dynamics of the Lactose Permease of Escherichia coli

描述（由申请人提供）：离子梯度耦合主动输运是一种生物现象，在健康和疾病中起着重要作用。多年来，该实验室一直专注于大肠杆菌的乳糖渗透酶（LacY）作为这类转运蛋白的范例。为了更详细地理解传输机制，需要原子水平上的结构以及时间分辨的动态信息。最近，LacY （Major Facilitator super family的一员，包含超过3000个成员）的内向构象的x射线结构在大约3.5 a时被解决。这种非常疏水的蛋白质由两个对称的6螺旋束组成，内部有一个巨大的亲水腔，其中包含糖结合位点，仅对细胞质开放。与此同时，在部分由DK 51131资助的情况下，Pi的实验室还构建并描述了LacY每个位置的单cys突变体库，以及其他位点定向突变体，以及一系列广泛使用的位点定向技术，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证据，已经提出了一种机制，其中包含结合位点的亲水腔可被膜的两侧交替访问。此外，质子电化学梯度已被证明通过加速限速步骤（质子解离）而不是改变膜两侧结合位点的亲和力来驱动糖的浓度梯度积累。因此，取得的进展使该实验室处于一个独特的位置，可以研究由LacY催化的转运的详细机制。在本研究中，我们将基于LacY的x射线结构，结合现有的各种生化和生物物理技术，使用合理设计的突变体来获得时间分辨的数据，这些数据将产生关于LacY的构象状态及其在配体结合和翻转过程中的相互转换的动态信息。

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.