Structural Characterization of the Na+/Glucose Cotransporter Family

描述(申请人提供)：活性转运蛋白参与多种细胞反应，促进特定分子通过包围所有细胞和细胞器的非渗透膜双层。这些完整的蛋白质为膜的功能奠定了基础，从而使所有形式的生命中能量的产生和必需营养的运输成为可能。此外，膜蛋白的功能异常与许多人类疾病有因果关系。因此，了解膜蛋白结构和功能的动力学是基础和医学研究的重要目标。在最初的资助周期中，我们解决了副溶血性弧菌Na+/半乳糖同向转运体(VSGLT)向内封闭构象的结构。最近，我们解决了vSGLT的一个向内开放的构象。总之，这些结构(结合生化和分子动力学模拟)表明，Na+的退出导致跨膜螺旋1的重新定位，从而打开了允许半乳糖释放的内部分子“门”。这一新的应用将利用在第一个周期中取得的成果，结合结晶学、最先进的光谱和漫反射X射线衍射技术来测量蛋白质整个区域的复杂运动。这些方法，加上生理分析和分子动力学模拟，将提供对膜运输的实时洞察力。这项建议有四个主要目标：1)我们将使用基本残基的抑制剂和突变体来改变vSGLT的构象平衡，并解析新的结构；2)我们将使用双电子-电子共振(DER)来监测底物诱导的构象变化；3)我们将使用时间分辨小角和广角X射线散射(TR-S/WAXS)来实时捕捉原子间的构象变化；以及4)我们将确定SSS家族中与药物相关的人类成员的结构。每个目标本身都能产生令人兴奋的结果，但当这些互补的方法合并在一起时，它们将提供更完整的Na+和糖共同运输的图景。 公共卫生相关性：活性运输蛋白参与多种细胞反应，是人类健康和疾病的重要组成部分。这些蛋白质的功能和动力学性质从根本上与它们的三维结构相耦合，这些三维结构在广泛的时间范围内受到原子水平的调节。这些研究将提供半乳糖转运体在其反应周期中进行的完整的原子拆分机制。

DESCRIPTION (provided by applicant): Active transport proteins are involved in a multitude of cellular reactions, facilitating the passage of specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. These integral proteins establish the basis for membrane function and thus make possible the generation of energy and transport of essential nutrients in all forms of life. Furthermore, aberrant function of membrane proteins is causally implicated in many human diseases. Understanding the dynamics of membrane protein structure and function therefore constitutes a critical objective for basic and medical research. During the initial grant cycle, we solved the structure of the Na+/galactose symporter from Vibrio parahaemolyticus (vSGLT) in the inward-occluded conformation. More recently, we solved an inward-open conformation of vSGLT. Together, these structures (in conjunction with biochemical and molecular dynamics simulations) show Na+ exit causes a reorientation of transmembrane helix 1 that opens an inner molecular "gate" permiting galactose release. This renewal application will capitalize on the gains made during the first cycle by combining crystallography, state-of-the-art spectroscopy and diffuse X-ray diffraction techniques to measure intricate movements of entire regions of the protein. These approaches, coupled with physiological assays and molecular dynamics simulations, will provide insights into membrane transport in real-time. This proposal has four primary goals: 1) we will use inhibitors and mutants of essential residues to alter the conformational equilibrium of vSGLT and resolve new structures; 2) we will monitor substrate-induced conformational changes using double electron-electron resonance (DEER); 3) we will capture real-time inter- atomic conformational changes using Time Resolved Small- and Wide-Angle X-ray Scattering (TR-S/WAXS); and 4) we will determine structures of the pharmaceutically relevant human members of the SSS family. Each aim on it own is capable of producing exciting results, but when these complementary approaches are merged together they will provide a more complete picture of Na+ and sugar co-transport. PUBLIC HEALTH RELEVANCE: Active transport proteins are involved in a multitude of cellular responses and important components in human health and disease. The functional and dynamical properties of these proteins are fundamentally coupled to their three-dimensional structures which are modulated at the atomic level over a broad range of time scales. These studies will provide a complete atomic resolution mechanism of the sodium galactose transporter as it proceeds through its reaction cycle.