Structural Studies of Sugar Transporters.

1.原项目总结 底物在细胞膜上的二次主动运输对许多细胞和 生理过程。最大的次级转运体家族是主要的促进者 超级家族(MFS)。甘油-3-磷酸(G3P)转运蛋白(GlpT) 大肠埃希菌膜是MFS的一员。在上一个资金周期中，我们 确定了GlpT的晶体结构，这表明底物移位的机制 穿过膜，这涉及到蛋白质的摇杆开关式运动。在.期间 当前的资金周期，我们的目标是了解转运蛋白的底物特异性，以测试 建议的运输机制，并表征所需的构象变化 底物运输。 2.桑迪带来的重大挫折 该项目的大部分原定目标已经实现，只有一部分除外 最初的目标4：获得外向型GlpT的晶体结构。部分内容 这个项目延迟完成是因为我们的机构受到了严重的破坏。 飓风桑迪。截至去年秋天，我们终于有了来自 日本慢生根瘤菌。由于该蛋白在GlpT同源物中排在第27位，我们 在寻找稳定的蛋白质进行结晶的过程中，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。因此， 我们18°C结晶室的温度升至48°C，导致我们所有的晶体都融化了。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.