Structural Characterization of the Na+/Glucose Cotransporter Family

描述（由申请人提供）：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不渗透膜双分子层。这些完整的蛋白质建立了膜功能的基础，从而使所有生命形式的能量产生和必需营养物质的运输成为可能。此外，膜蛋白的异常功能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构成了基础和医学研究的关键目标。在最初的资助周期中，我们解决了来自副溶血性弧菌（vSGLT）的内闭塞构象的Na+/半乳糖同调体的结构。最近，我们解决了vSGLT的内向开放构象。总之，这些结构（结合生化和分子动力学模拟）表明，Na+出口导致跨膜螺旋1重新定向，打开内部分子“门”，允许半乳糖释放。这一更新应用将利用第一个周期所取得的成果，结合晶体学、最先进的光谱学和漫射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，将提供实时膜运输的见解。本研究有四个主要目标：1)我们将利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（DEER）来监测衬底诱导的构象变化；3)我们将利用时间分辨小角和广角x射线散射（TR-S/WAXS）捕捉实时原子间构象变化；4)我们将确定与SSS家族相关的人类成员的结构。每个目标本身都能够产生令人兴奋的结果，但是当这些互补的方法合并在一起时，它们将提供Na+和糖共同运输的更完整的画面。

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.