Our work probes the structure and mechanism of a bacterial membrane transporter, OxlT, a member of the Major Facilitator Superfamily (MFS). Other members of the MFS play essential roles in human physiology, enabling the absorption of sugars and amino acids by most cells and the cycling of neurotransmitters by neurons in the central nervous system. None of these human examples can be studied in the detail possible with OxlT, so work with this model should be instructive as to (a) the various conformations adopted by transporters during substrate binding and transport and (b) how such transporters establish their substrate specificity. Such information should make it easier to both diagnose disease and design interventions for treatment. Three general lines of study are planned. (1) Preliminary work has established conditions yielding high level production of OxlT that is stable, monodisperse and of low lipid content. We will now pursue x-ray crystallography of OxlT with the advice and help of interested collaborators, beginning with manual and robotic-based screens centered on conditions that have already produced small crystals. (2) The structural analysis of OxlT will be paralleled by biochemical work focused on (a) determination of the OxlT oligomeric state in both soluble and membrane-embedded forms;(b) site-directed mutagenesis of residues lining the permeation pathway, so as to understand factors that set substrate specificity/selectivity;and (c) identification of residues that show substrate-triggered changes in proximity consistent with their role(s) in the opening and closing of the permeation pathway. (3) Our third initiative involves collaborative work to place pairs of fluorescent probes at strategic positions on OxlT and use single-pair fluorescence resonance energy transfer (spFRET) to measure separation distances changes at the single-molecule level. This will give a catalog of OxlT conformations adopted during substrate binding and transport and will link structural and kinetic models.
This work seeks to reveal the mechanism by which a transporter, OxlT, facilitates substrate movement across membranes. The work relates to human health, since OxlT closely resembles systems that move sugars in the gut and kidney, cycle neurotransmitters in the nervous system and confer drug resistance to pathogenic bacteria.
|Iyalomhe, Osigbemhe; Khantwal, Chandra M; Kang, Di Cody (2015) The Structure and Function of OxlT, the Oxalate Transporter of Oxalobacter formigenes. J Membr Biol 248:641-50|
|Iyalomhe, Osigbemhe; Herrick, Dawn Z; Cafiso, David S et al. (2014) Closure of the cytoplasmic gate formed by TM5 and TM11 during transport in the oxalate/formate exchanger from Oxalobacter formigenes. Biochemistry 53:7735-44|
|Lesoine, John F; Venkataraman, Prahnesh A; Maloney, Peter C et al. (2012) Nanochannel-based single molecule recycling. Nano Lett 12:3273-8|
|Kang, Di-Cody; Venkataraman, Prahnesh A; Dumont, Mark E et al. (2011) Oligomeric state of the oxalate transporter, OxlT. Biochemistry 50:8445-53|
|Wang, Xicheng; Ye, Liwen; McKinney, Caleb C et al. (2008) Cysteine scanning mutagenesis of TM5 reveals conformational changes in OxlT, the oxalate transporter of Oxalobacter formigenes. Biochemistry 47:5709-17|
|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|
|Wang, Xicheng; Sarker, Rafiquel I; Maloney, Peter C (2006) Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes. Biochemistry 45:10344-50|
|Yang, Qiang; Wang, Xicheng; Ye, Liwen et al. (2005) Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes. Proc Natl Acad Sci U S A 102:8513-8|
|Hall, Jason A; Maloney, Peter C (2005) Altered oxyanion selectivity in mutants of UhpT, the Pi-linked sugar phosphate carrier of Escherichia coli. J Biol Chem 280:3376-81|
|Fann, Mon-Chou; Busch, Anne; Maloney, Peter C (2003) Functional characterization of cysteine residues in GlpT, the glycerol 3-phosphate transporter of Escherichia coli. J Bacteriol 185:3863-70|
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