Structural Studies of Sugar Transporters.

1.原项目摘要 底物跨细胞膜的次级主动转运对许多细胞和生物学过程至关重要。 生理过程。最大的次级转运蛋白家族是主要的促进剂 超家族（MFS）。内分泌细胞的sn-甘油-3-磷酸（G3 P）转运蛋白（GlpT） 大肠杆菌的膜是MFS的成员。在上一个融资周期， 确定了GlpT晶体结构，这表明了底物移位的机制 这涉及蛋白质的摇杆开关式运动。期间 目前的资金周期，我们的目标是了解转运蛋白的底物特异性，测试 提出的运输机制，并表征所需的构象变化， 基质传输 2.由于桑迪， 该项目的大部分原始目标已经实现，除了一部分， 最初的目标4：获得GlpT的外向构象的晶体结构。的一部分 完成该项目的延误是由于我们的机构受到的损害， 飓风桑迪。截至去年秋天，我们终于有了4个来自 日本慢生根瘤菌由于该蛋白质在GlpT同源物中排名第27， 为了寻找稳定的结晶蛋白质，我们将其命名为G27蛋白。 然而，在飓风期间，这个校园失去了电力和冷却水。结果导致 我们18 °C结晶室的温度上升到48 °C，导致我们所有的晶体熔化。我们 目前正在尝试重复结晶实验。

1. Summary of the original project Secondary active transport of substrates across the cell membrane is crucial to many cellular and physiological processes. The largest secondary transporter family is the major facilitator superfamily (MFS). The sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli is a member of the MFS. In the previous funding cycle we determined the GlpT crystal structure, which suggests a mechanism for substrate translocation across the membrane that involves a rocker-switch-type movement of the protein. During the current funding cycle, we aimed to understand the transporter’s substrate specificity, to test the proposed transport mechanism, and to characterize the conformational changes needed for substrate transport. 2. Major setback due to Sandy Most of the original aims of the project were already accomplished, except one part of the original Aim 4: to get a crystal structure of GlpT in an outward-facing conformation. Part of the delay in finishing the project was due to the damage that was inflicted onto our institution by Hurricane Sandy. As of last fall, we finally had 4 Å crystals of a GlpT homolog from Bradyrhizobium japonicum. As this protein was number 27 among the GlpT homologs we screened for in our search for a stable protein for crystallization, we named it protein G27. However, during the hurricane, this campus lost its power and cooling water. As a result, the temperature in our 18 °C crystallization room rose to 48 °C, causing all our crystals to melt. We are currently trying to repeat the crystallization experiments.