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.

美国国立卫生研究院 1R01GM122759-01A1 膜转运蛋白转运机制的综合方法 项目概要/摘要 我们的长期目标是了解阳离子/溶质同构催化的分子机制 这些转运蛋白在维持正常细胞活动中发挥着关键作用，非常重要。 在人类健康和疾病中，可以作为药物靶点和治疗传递途径。 根据提案，我们计划研究细菌 Na+ 偶联蜜二糖渗透酶 (MelB)，它利用储存的能量 在 Na+、Li+ 或 H+ 的电化学梯度中驱动半乳糖苷相对于其的易位 浓度梯度，是探索 MFS 家族同向转运蛋白分子机制的原型 可以使用一种以上的阳离子物种进行偶联，在血脑中表达的 MelB 同系物。 血视网膜屏障催化 Na+ 偶联的二十二碳六烯酸 (DHA) 摄取 溶血磷脂酰胆碱 (LPC)，为大脑和眼睛提供必需的 DHA，促进神经发育和 一般来说，对于次级主动运输来说，驱动之间的耦合。 阳离子和货物溶质是强制性的，但能量耦合背后的机制在很大程度上仍然存在 我们将通过组合方法阐明 Na+ 偶联的同向机制，包括。 遗传学、生物化学、量热法、连续波电子定点自旋标记 (SDSL) 我们创建了高顺磁共振波谱 (CW-EPR) 和 3-D X 射线晶体学。 亲和力 MelB-camelid 单域纳米抗体 (Nbs) 用于 MelB 结晶，并已实现 等温滴定量热法 (ITC) 测量以确定自由能变化和热容量 MelB 配体（蜜二糖、Na+、Li+）单独或一起的结合以及 CW-EPR 的变化 基于我们强有力的初步测量，测量配体诱导的溶剂可及性变化和接近度变化。 结果，提出了三个独立但互补的目标来检验我们的中心假设： 共转运机制是共底物的协同结合，诱导形成封闭的 我们的综合多学科方法将提供重要的缺失信息。 阳离子/溶质同向机制并提高我们对配体结合的基础知识 一般的能量学和蛋白质构象变化，以及对 Na+- 其他研究的直接影响 耦合转运蛋白，包括大脑和视网膜中的 LPC 转运蛋白。