Crystallization of eukaryotic facilitated glucose transporters

描述(由申请人提供)：本项目的长期目标是通过结合X射线结晶学和生化/生物物理方法，了解主要促进剂超家族(MFS)中具有重要生理和临床意义的人促进葡萄糖转运蛋白(GLUT)的结构和功能。Glut1在人类红细胞(RBC)和血脑屏障中含量丰富，是这些易化转运蛋白的广泛研究代表。GLUT4负责胰岛素调节的葡萄糖处置。已发现几种疾病具有导致谷氨酸功能障碍的突变，如GLUT1缺乏综合症和II型糖尿病。重要的是要获得高分辨率的结构，以及在原子水平上表征传输机制。到目前为止，还没有可用于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。蛋白质的生产和结晶是结构测定的两大瓶颈。这项应用的等电点已经证明，操纵磷脂(PL)可以改善来自大肠杆菌的乳糖渗透酶晶体的质量，这是MFS的典范，以及其他几种疏水性膜蛋白。为了验证PL和/或中性脂在人类GLUT促进剂结晶过程中起重要作用的假设，PI优化了一种简单的方法，通过从过时的人红细胞制备的RBC鬼膜中差异分离来获得Glut1。此外，还建立了获得人Glut1和GLUT4在酿酒酵母中过表达的专用系统。这一应用的具体目的包括：1)通过重组表达和差异分离来优化蛋白质生产，以提高产量和纯度。将筛选不同位置的不同亲和标签，用于表达、纯化、稳定性和功能。2)关于晶体质量的PL和中性脂的表征。系统地测试磷脂和/或中性脂对功能、稳定性以及晶体质量的重要作用(S)。人们的注意力将集中在天然脂质上。3)优化了用于X射线结构测定的晶化程序。任何谷氨酸的晶体结构都将有助于我们对转运机制的理解，并为合理的新药设计和治疗干预奠定基础。此外，该结构还有望为确定脂类在膜蛋白结晶中的作用提供有价值的信息。相关声明：真核细胞葡萄糖的成功结晶对于获得X射线晶体结构是必不可少的；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。表征磷脂或中性脂在谷蛋白结晶中的作用(S)将有助于建立新方法在其他膜蛋白结晶中应用的基本指导方针。

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.