Integrated approaches to symport mechanisms of membrane transporters

膜转运蛋白转运机制的综合方法

• 批准号: 9895210
• 负责人: Lan Guan
• 金额: $ 4.42万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895210
• 关键词: 3-Dimensional; Active Biological Transport; Affinity; Automobile Driving; Binding; Biochemistry; Blood; Brain; Calorimetry; Cations; Coupled; Coupling; Crystallization; Disease; Docosahexaenoic Acids; Drug Targeting; Electron Spin Resonance Spectroscopy; Eye; Family; Free Energy; Galactosides; Genetic; Health; Homologous Gene; Human; Ion Cotransport; Knowledge; Ligand Binding; Ligands; Lysophosphatidylcholines; Measurement; Measures; Membrane; Membrane Transport Proteins; Molecular; Nerve Degeneration; Neurodegenerative Disorders; Pathway interactions; Play; Prevention; Protein Conformation; Retina; Role; Site; Solvents; Spin Labels; Structure; Testing; Titrations; United States National Institutes of Health; X-Ray Crystallography; base; improved; interdisciplinary approach; melibiose permease; nanobodies; neurodevelopment; novel; novel therapeutics; prevent; prototype; solute; success; symporter; targeted treatment; uptake

NIH 1R01GM122759-01A1 Integrated approaches to symport mechanisms of membrane transporters PROJECT SUMMARY/ABSTRACT Our long-term objective is to understand the molecular mechanisms of cation/solute symport catalyzed by membrane carriers. These transporters play critical roles in maintaining normal cellular activities, are important in human health and disease, and can serve as drug targets and therapeutic delivery pathways. In this proposal, we plan to study the bacterial Na+-coupled melibiose permease (MelB), which utilizes energy stored in the electrochemical gradient of Na+, Li+, or H+ to drive the translocation of galactoside against its concentration gradient, and is a prototype for exploring molecular mechanisms of symporters in the MFS family that can use more than one cationic species for coupling. The MelB homologue expressed in blood-brain and blood-retina barriers catalyzes Na+-coupled uptake of docosahexaenoic acids (DHA)-carrying lysophosphatidylcholine (LPC), thus supplying essential DHA to brain and eyes for neural development and prevents neurodegeneration. For secondary-active transport in general, the coupling between the driving cation and cargo solute is obligatory, but the mechanisms underlying the energetic coupling remain largely unknown. We will elucidate the Na+-coupled symport mechanisms by a combined approach, including genetics, biochemistry, calorimetry, site-directed spin labeling (SDSL) with continuous-wave electron paramagnetic resonance spectroscopy (CW-EPRs), and 3-D X-ray crystallography. We have created high- affinity MelB-camelid single-domain nanobodies (Nbs) for crystallization of MelB, and have implemented isothermal titration calorimetry (ITC) measurements to determine the free-energy changes and heat capacity changes for the binding of MelB’s ligands (melibiose, Na+, Li+), alone or together, as well as the CW-EPRs to measure ligand-induced solvent accessibility changes and proximity changes. Based on our strong preliminary results, three independent but complementary aims are proposed to test our central hypothesis: the core of the symport mechanism is cooperative binding of co-substrates that induces the formation of an occluded intermediate state. Our integrated multi-disciplinary approach will provide important missing information into the cation/solute symport mechanisms and improve our fundamental knowledge of the ligand binding energetics and protein conformational changes in general, as well as directly impact on other studies of Na+- coupled transporters including the LPC transporter in brain and retina.

NIH 1R01GM122759-01A1 综合方法的膜转运蛋白交通机理 项目摘要/摘要 我们的长期目的是了解阳离子/溶质的分子机制由 膜载体。这些转运蛋白在维持正常的细胞活性中起关键作用，很重要 在人类健康和疾病中，可以用作药物靶标和治疗性递送途径。在这个 提案，我们计划研究利用存储的能量的细菌Na+偶联的粉状粘液（MELB） 在Na+，Li+或H+的电化学梯度中，驱动半乳糖苷的易位 浓度梯度，是探索MFS家族分子机制的原型 可以使用多个阳离子物种进行耦合。梅尔布同源物在血脑和 血液 - 逆转障碍催化Na+耦合摄入docosahexaenoic酸（DHA） - 携带 溶物磷脂酰胆碱（LPC），因此为脑和眼睛提供必不可少的DHA，以进行神经元发育和 防止神经变性。对于一般的二级运输，驾驶之间的耦合 阳离子和货物可溶性是强制性的，但是能量耦合的基础机制在很大程度上仍然保持 未知。我们将通过合并方法阐明NA+耦合的交构机制，包括 具有连续波电子的遗传学，生物化学，量热法，定向旋转标签（SDSL） 顺磁共振光谱（CW-EPR）和3-D X射线晶体学。我们创建了高 - 亲和力melb-camelid单域纳米型（NB）用于结晶，并已实施 等温滴定量热法（ITC）测量值，以确定自由能的变化和热容量 梅尔布配体（Melibiose，Na+，Li+）的结合，单独或一起的结合以及CW-EPR与 测量配体诱导的溶剂可及性的变化和接近性变化。基于我们强大的初步 结果，提出了三个独立但完整的目标来检验我们的中心假设： 交易机制是诱导封闭形成的共缩合物的合作结合 中间状态。我们综合的多学科方法将为 阳离子/溶质的交配机制，并提高我们对配体结合的基本知识 一般而言，能量和蛋白质构象变化，以及直接影响Na+的其他研究 耦合转运蛋白包括大脑和视网膜中的LPC转运蛋白。