Integrated approaches to symport mechanisms of membrane transporters

膜转运蛋白联合运输机制的综合研究 项目摘要/摘要 我们的长期目标是了解阳离子/溶质共转运的分子机制。 膜载体。这些转运蛋白在维持正常的细胞活动中起着关键作用，是重要的 在人类健康和疾病中发挥重要作用，可作为药物靶点和治疗给药途径。在这 建议，我们计划研究细菌钠偶联蜜糖渗透酶(Melb)，它利用储存的能量 在Na、Li或H的电化学梯度中，以驱动半乳糖苷对其 浓度梯度，是探索MFS家族中共转运蛋白分子机制的原型 可以使用不止一种阳离子物种进行偶联。在血脑中表达的Melb同源物和 血-视网膜屏障催化携带二十二碳六烯酸(DHA)的钠偶联摄取 溶血磷脂酰胆碱(LPC)，从而为大脑和眼睛提供必要的DHA，促进神经发育和 防止神经退化。对于二次主动运输来说，驾驶之间的耦合 阳离子和货物溶质是必需的，但能量耦合的机制在很大程度上仍然存在 未知。我们将通过一种组合的方法来阐明钠偶联的符号传递机制，包括 遗传学、生物化学、量热、连续波电子定点自旋标记(SDSL) 顺磁共振波谱(CW-EPRS)和三维X射线结晶学。我们创造了高- 亲和Melb-Camelid单结构域纳米体(NBS)用于Melb的结晶，并已实现 等温滴定量热法(ITC)测定自由能变化和热容 Melb的配体(Melibiose，Na，Li)单独或共同结合的变化，以及CW-EPs对 测量配体诱导的溶剂可及性变化和邻近性变化。基于我们强有力的初步调查 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： 共载体机制是共底物的协同结合，它诱导形成被遮挡的 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/溶质共输送机制和提高我们对配基结合的基础知识 一般的能量学和蛋白质构象变化，以及直接影响其他研究的钠- 脑和视网膜中的偶联转运蛋白，包括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.