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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
3R01DK053973-12S1
Application #
8663524
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Sechi, Salvatore
Project Start
2014-01-10
Project End
2015-12-31
Budget Start
2014-01-10
Budget End
2015-12-31
Support Year
12
Fiscal Year
2014
Total Cost
$113,679
Indirect Cost
$46,612
Name
New York University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
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
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
Czyzewski, Bryan K; Wang, Da-Neng (2012) Identification and characterization of a bacterial hydrosulphide ion channel. Nature 483:494-7
Mancusso, Romina; Karpowich, Nathan K; Czyzewski, Bryan K et al. (2011) Simple screening method for improving membrane protein thermostability. Methods 55:324-9
Karpowich, Nathan K; Wang, Da-Neng (2010) Biophysics: Transporter in the spotlight. Nature 465:171-2
Law, Christopher J; Enkavi, Giray; Wang, Da-Neng et al. (2009) Structural basis of substrate selectivity in the glycerol-3-phosphate: phosphate antiporter GlpT. Biophys J 97:1346-53
Karpowich, Nathan K; Wang, Da-Neng (2008) Structural biology. Symmetric transporters for asymmetric transport. Science 321:781-2
Law, Christopher J; Almqvist, Jonas; Bernstein, Adam et al. (2008) Salt-bridge dynamics control substrate-induced conformational change in the membrane transporter GlpT. J Mol Biol 378:828-39
Law, Christopher J; Maloney, Peter C; Wang, Da-Neng (2008) Ins and outs of major facilitator superfamily antiporters. Annu Rev Microbiol 62:289-305
Law, Christopher J; Yang, Qiang; Soudant, Celine et al. (2007) Kinetic evidence is consistent with the rocker-switch mechanism of membrane transport by GlpT. Biochemistry 46:12190-7

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