Dynamics of the Lactose Permease of Escherichia coli

描述(申请人提供)：离子梯度耦合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，该实验室一直将重点放在大肠杆菌的乳糖渗透酶(Lacy)上，以此作为这类运输蛋白的范例。为了更详细地了解传输机制，需要原子级别的结构以及时间分辨的动态信息。最近，包含3000多个成员的主要促进器超家族的成员Lacy的内向构象的X射线结构在大约3.5A被解析出来。这种非常疏水的蛋白质由两个对称的6-螺旋束组成，具有一个巨大的亲水内腔，其中包含糖结合部位，并且只对细胞质开放。与此同时，在51131丹麦克朗的部分资助下，PI的实验室还在Lacy的每个位置构建了一个单半胱氨酸突变体文库，并对其进行了鉴定，以及其他定点突变体，以及一系列可轻松应用于动态研究的广泛使用的定点突变技术。基于这种结构和大量的生化和生物物理证据，提出了一种机制，即包含结合位点的亲水空腔可以交替地到达膜的两侧。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部位的亲和力来推动糖的积累。因此，所取得的进展使该实验室处于独特的地位，可以研究花边催化的详细运输机制。在这项建议中，我们将使用基于Lacy的X射线结构的合理设计的突变体，结合现有的各种生化和生物物理技术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周转期间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.