Crystallization of eukaryotic facilitated glucose transporters

描述(申请人提供)：这个项目的长期目标是通过结合X射线结晶学和生化-生物物理方法来了解生理和临床上重要的膜转运蛋白的结构和功能。膜蛋白在细胞功能的许多方面起着至关重要的作用；一些膜蛋白是药理和毒理活性物质的靶标。人促进葡萄糖转运蛋白(Gluts)是主要促进剂超家族(MFS)的成员，由一个相对较大的结构相关蛋白家族(GLUT1-12)组成。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。已发现几种疾病与谷氨酸的突变和功能障碍有关，例如GLUT1缺乏综合症，一种遗传性神经综合征，以及II型糖尿病，这是对人类健康的主要威胁之一，其流行规模正变得令人震惊。因此，获得高分辨率的结构以及在分子水平上表征其传输机制是非常重要的。到目前为止，还没有可用于任何葡萄糖促进剂的3D晶体结构，尽管已经进行了许多尝试，特别是Glut1。这项建议将集中在真核促进谷氨酸的结晶，特别是来自人红细胞的Glut1。从人红细胞中分离纯化Glut1已有二十多年的历史。最近，该提案的等电点观察到，磷脂(PL)在MFS成员大肠杆菌乳糖渗透酶的结晶过程中起着重要作用。操纵PL极大地提高了我们提取结晶相关膜蛋白的成功率。因此，将荧光用于结晶将为获得MFS的真核成员的晶体结构提供一种很有前途的方法。将从红细胞膜中提纯MFS的人类蛋白Glut1；将同时测试真核细胞Glut1-4在不同系统中的过度表达系统。除了常规的结晶方法外，还将引入控制PL含量的方法来结晶人Glut1和/或真核细胞谷蛋白，这将为未来解决这些重要的真核转运蛋白的X射线晶体结构奠定基础。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；预期的结构将极大地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。

DESCRIPTION (provided by applicant): The long term goals of this project are to understand the structure and function of physiologically and clinically important membrane transporters through a combination of X-ray crystallography and biochemical-biophysical approaches. Membrane proteins play crucial roles in many aspects of cell function; some are targets for pharmacologically and toxicologically active substances. The human facilitated glucose transporters (Gluts), members of the Major Facilitator Superfamily (MFS), comprise a relatively large family of structurally related proteins (GLUT1-12). 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 the mutation and malfunction of Gluts, such as GLUT1 deficiency syndrome, a hereditary neurological syndrome, and type II diabetes, one of major threats to human health that is becoming alarmingly epidemic in scale. Therefore, it is important to obtain a high-resolution structure, as well as characterize the transport mechanism at a molecular level. So far, there is no 3-D crystal structure available for any glucose facilitator, although many attempts have been made, particularly with Glut1. This proposal will focus on the crystallization of eukaryotic facilitated Gluts, particularly Glut1 from human RBCs. Isolation and purification of Glut1 from human RBCs was achieved more than two decades ago. Recently, the PI of this proposal observed that phospholipids (PL) play an important role in the crystallization of lactose permease of Escherichia coli, a member of MFS. Manipulating PL has dramatically improved our success rate for crystallization related membrane proteins. Therefore, the use of PL in crystallization should yield a promising approach for obtaining crystal structures of eukaryotic members of the MFS. Glut1, a human protein of MFS, will be purified from RBC membranes; over-expression systems of eukaryotic Glut1-4 in different systems will be simultaneously tested. Manipulation of PL content will be introduced in addition to conventional crystallization methods, to crystallize human Glut1 and/or the eukaryotic Gluts, which will lay the foundation for solving X-ray crystal structures of these important eukaryotic transporters in the future. 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.