Crystallization of eukaryotic facilitated glucose transporters

描述（由申请人提供）：该项目的长期目标是通过x射线晶体学和生物化学/生物物理方法的结合，了解生理和临床重要的人类促进葡萄糖转运蛋白（Gluts）的结构和功能，这些转运蛋白是主要促进物超家族（MFS）的成员。Glut1在人红细胞（rbc）和血脑屏障中含量丰富，是这些促进转运体的一个广泛研究的代表。Glut4负责胰岛素调节的葡萄糖处理。一些疾病已被确定与突变导致GLUT1功能障碍，如GLUT1缺乏症和II型糖尿病。重要的是获得高分辨率的结构，以及在原子水平上表征输运机制。到目前为止，还没有任何葡萄糖促进剂的三维晶体结构，尽管已经进行了许多尝试，特别是Glut1。蛋白质的产生和结晶是结构测定的两个主要瓶颈。本应用程序的PI已经证明，操纵磷脂（PL）可以提高大肠杆菌乳糖渗透酶晶体的质量，这是MFS和其他几种疏水膜蛋白的范例。为了验证PL和/或中性脂类在人类Glut促进剂结晶过程中发挥重要作用的假设，PI优化了一种简单的方法，通过从过时的人红细胞制备的红细胞鬼膜中分离得到Glut1。此外，还建立了一个专门的系统来获得人Glut1和Glut4在酿酒sacroomyces cerevisiae中的过表达。该应用程序的具体目的包括：1)通过重组表达和差异分离优化蛋白质生产，以增加数量和纯度。每个Glut在不同位置的不同亲和标签将被筛选表达、纯化、稳定性和功能。2) PL和中性脂在晶体质量方面的表征。PL和/或中性脂在功能、稳定性和晶体质量方面的重要作用将被系统地测试。注意力将集中在天然脂质上。3) x射线结构测定结晶工艺的优化。研究glut的晶体结构将对我们理解转运机制有重要意义，并为合理设计新药和治疗干预奠定基础。此外，该结构也有望为定义脂质在膜蛋白结晶中的作用提供有价值的信息。相关声明：真核Gluts的成功结晶是获得x射线晶体结构的必要条件；预期的结构将大大提高我们对促进葡萄糖转运的理解，并为治疗干预提供重要线索，这将在生物学和医学上产生重大影响。研究PL或中性脂类在Gluts结晶过程中的作用，将为该方法在其他膜蛋白结晶过程中的应用奠定基础。

DESCRIPTION (provided by applicant): The long term goals of this project are to understand the structure and function of physiologically and clinically important human facilitated glucose transporters (Gluts), members of the Major Facilitator Superfamily (MFS), through a combination of X-ray crystallography and biochemical/biophysical approaches. Glut1, which is abundant in the human red blood cells (RBCs) and the blood-brain barrier, is an extensively-studied representative of these facilitated transporters. Glut4 is responsible for insulin-regulated glucose disposal. Several diseases have been identified with mutations resulting in malfunction of Gluts, such as GLUT1 deficiency syndrome and type II diabetes. It is important to obtain a high-resolution structure, as well as characterize the transport mechanism at an atomic level. Thus far, there is no 3-D crystal structure available for any glucose facilitator, although many attempts have been made, particularly with Glut1. Protein production and crystallization are two major bottlenecks for the structural determination. The PI of this application has demonstrated that manipulating phospholipids (PL) improves the quality of crystals of the lactose permease from Escherichia coli, a paradigm for the MFS, and several other hydrophobic membrane proteins. To test the hypothesis that PL and/or neutral lipids play an important role in crystallization of human Glut facilitators, the PI has optimized a simple method to obtain Glut1 by differential isolation from RBC ghost membranes, which are prepared from out-dated human RBCs. Furthermore, a dedicated system to obtain the overexpression of human Glut1 and Glut4 in Sachromyces cerevisiae has also been achieved. Specific aims of this application include: 1) Optimization of protein production by both recombinant expression and differential isolation to increase quantity and purity. Different affinity tags at different locations with each Glut will be screened for expression, purification, stability and function. 2) Characterization of PL and neutral lipids with respect to crystal quality. The important role(s) of PL and/or neutral lipids for function, stability, as well as crystal quality will be systematically tested. Attention will be focus on the native lipids. 3) Optimization of a crystallization procedure for X-ray structure determination. Crystal structure of any Gluts will significantly contribute to our understanding of transport mechanism and lay the foundation for rational new drug design and therapeutic intervention. Furthermore, the structure is also expected to provide valuable information in defining the role of lipids in crystallization of membrane proteins. Relevance Statement: Successful crystallization of eukaryotic Gluts is imperative to obtaining X-ray crystal structures; the expected structures will substantially improve our understanding of facilitated glucose transport and provide important clues for therapeutic intervention, which will have significant impact in biology and medicine. Characterization of role(s) of PL or neutral lipids in crystallization of Gluts will help in the establishing of a basic guideline for application of the novel approach in other membrane protein crystallization.