Dynamics of the Lactose Permease of Escherichia Coli

大肠杆菌乳糖渗透酶的动力学

• 批准号: 9355287
• 负责人: Howard Ronald KABACK
• 金额: $ 9.53万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9355287
• 关键词: ATP-Binding Cassette Transporters; Affinity; Bacteria; Belief; Binding; Binding Sites; Biochemical; Cells; Collaborations; Complex; Coupled; Crystallization; Data; Detergents; Diabetes Mellitus; Disease; Dissociation; Drug Prescriptions; Electrons; Enzymes; Epilepsy; Epithelium; Escherichia coli; Exhibits; Family; Fluorescence; Fluorescent Probes; G-Protein-Coupled Receptors; Galactosides; Health; Human; Individual; Ion Cotransport; Kinetics; Label; Laboratories; Lactose; Lead; Ligand Binding; Ligands; Maps; Membrane; Membrane Proteins; Membrane Transport Proteins; Mental Depression; Methodology; Methods; Modeling; Molecular Conformation; Multi-Drug Resistance; Organism; Pharmacologic Substance; Physiology; Protein Dynamics; Proteins; Proton Pump Inhibitors; Protons; Research; Resolution; Roentgen Rays; SLC2A1 gene; San Francisco; Selective Serotonin Reuptake Inhibitor; Side; Site; Stomach; Structure; Testing; Time; Transmembrane Transport; base; clinically significant; conformational conversion; deprotonation; genome sequencing; glucose transport; improved; innovation; lactose permease; member; mutant; nanobodies; professor; proteoliposomes; protonation; reconstitution; research study; sugar; symporter; synaptic function; tool; vesicular monoamine transporter

ABSTRACT The aim of this proposal is to develop an atomic-level understanding of the mechanism of lactose/H+ symport by the lactose permease of Escherichia coli (LacY), an important model for the Major Facilitator Superfamily (MFS), the largest family of membrane transport proteins. The MFS contains many members that have clinical significance such as the vesicular monoamine transporter, as well as the GLUTs, which facilitate glucose transport into many different cells in the body. Like channels and ABC transporters, symporters are also highly relevant to human health (e.g. transport across epithelia, synaptic function) and disease (e.g. depression, epilepsy, diabetes, multidrug resistance). Also notable, at least two of the most widely used pharmaceuticals in the world [serotonin selective reuptake inhibitors (SSRIs) and gastric proton pump inhibitors (PPIs)] are targeted to membrane transport proteins. Also important, the innovative methods developed by the PI to study LacY have been applied directly to such important human membrane proteins as GLUT1 and G-Protein-Coupled Receptors (GPCRs). However, despite a number of structures of MFS members, including 7 of LacY, the mechanism of this dynamic protein is not completely understood. It has been demonstrated that sugar binding to highly dynamic protonated LacY triggers a global conformational change in which sugar- and H+-binding sites gain alternating access to either side of the membrane. Sugar binding and dissociation drive this conformational change through an induced-fit mechanism, while the proton electrochemical gradient enhances the rate of deprotonation. Therefore, LacY behaves much like an enzyme except that the transition state(s) involves the protein rather than the substrate. X-ray structures of LacY inward- and almost occluded outward-facing conformations provide the structural basis for studying the alternating access mechanism. The alternating access mechanism has been documented unequivocally by applying pre-steady state kinetics, as well as multiple biochemical and spectroscopic approaches pioneered in this laboratory, and by using kinetic data obtained in real time for several steps in the transport cycle. Thirty-one Camelid nanobodies now in our possession allow stabilization of LacY in different intermediate states that will provide an in-depth understanding of structural changes underlying the symport mechanism. It should be emphasized that the approaches and the methodologies described here for LacY are already being applied to other membrane transport proteins and can be applied to membrane proteins in general.

摘要 该提案的目的是发展对乳糖/H+机制的原子水平理解 大肠杆菌乳糖渗透酶（LacY）的共转运，这是主要促进剂的重要模型 超家族（MFS）是最大的膜转运蛋白家族。MFS包含许多成员 具有临床意义的蛋白质，如囊泡单胺转运蛋白，以及GLUT， 促进葡萄糖转运到体内许多不同的细胞中。像频道和ABC传输器一样， 协同转运蛋白也与人类健康高度相关（例如跨上皮细胞的转运，突触功能） 和疾病（例如抑郁症、癫痫、糖尿病、多药耐药性）。同样值得注意的是，至少有两个 世界上最广泛使用的药物[5-羟色胺选择性再摄取抑制剂（SSRIs）和胃 质子泵抑制剂（PPI）]靶向膜转运蛋白。此外，创新 PI开发的研究LacY的方法已直接应用于这些重要的人类 GLUT 1和G蛋白偶联受体（GPCR）。然而，尽管一些 MFS成员的结构，包括7个LacY，这种动态蛋白的机制不是 完全理解已经证明，糖与高度动态质子化的LacY结合， 引发了一个全球性的构象变化，其中糖和H+结合位点获得交替访问， 膜的两侧。糖的结合和解离通过一个分子链驱动这种构象变化。 诱导拟合机制，而质子电化学梯度提高去质子化速率。 因此，LacY的行为很像酶，除了过渡态涉及蛋白质 而不是衬底。LacY向内和几乎闭塞向外的X射线结构 构象为研究交替进入机制提供了结构基础。交替 访问机制已被明确记录通过应用前稳态动力学，以及 本实验室开创了多种生物化学和光谱学方法，并利用动力学数据 在运输周期的几个步骤中以真实的时间获得。31骆驼纳米抗体现在在我们的 拥有允许LacY稳定在不同的中间状态，这将提供一个深入的 了解结构变化的基础symport机制。应当强调， 这里描述的用于LacY的方法和方法已经被应用于其他膜。 转运蛋白，并且通常可以应用于膜蛋白。