Integrated approaches to symport mechanisms of membrane transporters

膜转运蛋白共转运机制的综合研究 项目总结/摘要 我们的长期目标是了解催化阳离子/溶质同向转运的分子机制， 膜载体这些转运蛋白在维持正常细胞活动中起关键作用， 在人类健康和疾病中，并且可以用作药物靶标和治疗递送途径。在这 建议，我们计划研究细菌Na+偶联蜜二糖通透酶（MelB），它利用储存的能量 在Na+、Li+或H+的电化学梯度中，驱动半乳糖苷的易位， 浓度梯度，是探索MFS家族中共转运蛋白分子机制的原型 其可以使用多于一种阳离子物质用于偶联。MelB同源物在血脑中表达， 血-视网膜屏障催化携带二十二碳六烯酸（DHA）的Na+偶联摄取 溶血磷脂酰胆碱（LPC），从而为大脑和眼睛提供神经发育所必需的DHA， 防止神经退化对于一般的二次主动输运， 阳离子和货物溶质是强制性的，但能量耦合的机制仍然很大程度上 未知我们将阐明Na+耦合的同向转运机制的综合方法，包括 遗传学、生物化学、量热法、连续波电子定点自旋标记（SDSL） 顺磁共振光谱（CW-EPR）和3-D X射线晶体学。我们创造了伟大的， 亲合MelB-骆驼科单域纳米抗体（Nbs）用于MelB的结晶，并已实施 等温滴定量热法（ITC）测量，以确定自由能变化和热容 MelB的配体（蜜二糖、Na+、Li+）单独或一起以及CW-EPR与 测量配体诱导的溶剂可及性变化和邻近性变化。根据我们初步的证据 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，其诱导形成封闭的 中间状态我们的综合多学科方法将提供重要的缺失信息， 阳离子/溶质同向转运机制，提高了我们对配体结合的基础知识 能量学和蛋白质构象变化的一般，以及直接影响其他研究的Na+- 偶联转运蛋白，包括脑和视网膜中的LPC转运蛋白。

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.