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 transporters 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 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.
| Karpowich, Nathan K; Song, Jinmei; Wang, Da-Neng (2016) An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin. J Mol Biol 428:3118-30 |
| Sauer, David B; Karpowich, Nathan K; Song, Jin Mei et al. (2015) Rapid Bioinformatic Identification of Thermostabilizing Mutations. Biophys J 109:1420-8 |
| Karpowich, Nathan K; Song, Jin Mei; Cocco, Nicolette et al. (2015) ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism. Nat Struct Mol Biol 22:565-71 |
| Mulligan, Christopher; Fitzgerald, Gabriel A; Wang, Da-Neng et al. (2014) Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae. J Gen Physiol 143:745-59 |
| Waight, Andrew B; Czyzewski, Bryan K; Wang, Da-Neng (2013) Ion selectivity and gating mechanisms of FNT channels. Curr Opin Struct Biol 23:499-506 |
| Karpowich, Nathan K; Wang, Da-Neng (2013) Assembly and mechanism of a group II ECF transporter. Proc Natl Acad Sci U S A 110:2534-9 |
| Loew, Leslie M; Wang, Da-Neng (2013) Science communication: Quality at stake. Science 342:1169 |
| Wang, Da-Neng; Stieglitz, Heather; Marden, Jennifer et al. (2013) Benjamin Franklin, Philadelphia's favorite son, was a membrane biophysicist. Biophys J 104:287-91 |
| Mancusso, Romina; Gregorio, G Glenn; Liu, Qun et al. (2012) Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter. Nature 491:622-6 |
| Czyzewski, Bryan K; Wang, Da-Neng (2012) Identification and characterization of a bacterial hydrosulphide ion channel. Nature 483:494-7 |
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