Structural Characterization of the Na+/Glucose Cotransporter Family

Na/葡萄糖协同转运蛋白家族的结构表征

• 批准号: 8333974
• 负责人: Jeffrey S Abramson
• 金额: $ 37.08万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8333974
• 关键词: Active Biological Transport; Basic Science; Behavior; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Carrier Proteins; Cell Communication; Cells; Coupled; Crystallization; Crystallography; Cysteine; Cytoplasm; DNA Sequence Rearrangement; Data; Diffuse; Disease; Electrons; Engineering; Environment; Equilibrium; Family; Galactose; Generations; Glucose; Goals; Grant; Health; Homologous Gene; Human; Iodides; Knowledge; Label; Life; Ligands; Maps; Measures; Medical Research; Membrane; Membrane Proteins; Methods; Molecular; Molecular Conformation; Monitor; Movement; Nutrient; Organelles; Pharmacologic Substance; Physiological; Positioning Attribute; Process; Property; Protein Region; Proteins; Pump; Reaction; Reagent; Resolution; Roentgen Rays; Series; Side; Sodium; Spectrum Analysis; Spin Labels; Structure; Techniques; Time; Transmembrane Transport; Vibrio parahaemolyticus; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; conformational conversion; design; drug development; human disease; inhibitor/antagonist; insight; member; molecular dynamics; mutant; novel; periplasm; protein structure function; research study; response; sugar; symporter; three dimensional structure; time use

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.

描述（由申请人提供）：主动转运蛋白参与了多种细胞反应，促进了特定分子跨越围绕所有细胞和细胞器的原本不可渗透的膜双层。这些整体蛋白为膜功能建立了基础，从而使能量的产生和各种形式的基本营养物质的运输产生。此外，膜蛋白的异常功能与许多人类疾病有关。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。 在最初的赠款周期中，我们从内向锁定构象中从颤动溶血症（VSGLT）中求解了Na+/半乳糖分类器的结构。最近，我们解决了VSGLT的内部开放构象。共同，这些结构（与生化和分子动力学模拟结合）显示了Na+出口会导致跨膜螺旋1的重新定位，从而打开了允许半乳糖释放的内部分子“栅极”。这种更新的应用将通过结合晶体学，最新光谱和弥漫性X射线衍射技术来衡量蛋白质整个区域的复杂运动，从而利用在第一个循环中所取得的收益。这些方法，再加上生理测定和分子动力学模拟，将为实时提供对膜运输的见解。该提案有四个主要目标：1）我们将使用基本残基的抑制剂和突变体改变VSGLT的构象平衡并解决新结构； 2）我们将使用双电子电子共振（DEER）监测底物诱导的构象变化； 3）我们将使用时间分辨的小角度和广角X射线散射（TR-S/WAXS）捕获实时原子间构象变化； 4）我们将确定SSS家族的药物相关的人类成员的结构。每个目标都可以产生令人兴奋的结果，但是当这些互补方法合并在一起时，它们将提供NA+和Sugar Co-Transport的完整图片。