Transporter Assays Using Solid Supported Membranes: A Novel Screening Platform for Drug Discovery

Kelety, Bela; Diekert, Kerstin; Tobien, Joanna; Watzke, Natalie; Dörner, Wolfgang; Obrdlik, Petr; Fendler, Klaus

doi:10.1089/adt.2006.4.575

Cited by 43 publications

(44 citation statements)

References 25 publications

(33 reference statements)

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“…When an electrochemical proton gradient was generated, uptake via DtpB as well as via DtpA proceeded until the gradient was collapsed by adding a protonophore. Moreover, capacity-coupled currents obtained with immobilized proteoliposomes on the SURFE 2 R one chip show the electrogenic nature of the DtpB-and DtpA-mediated transport process, similar to on-chip recordings with membranes containing the mammalian PEPT transporters [29]. This unequivocally establishes that dipeptide transport by the two bacterial transporters occurs by H + -symport.…”

Section: Transport Modesupporting

confidence: 67%

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton‐dependent peptide transporters of Escherichia coli

et al. 2008

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Uptake of peptides into Escherichia coli cells is thought to be mediated by three different transport systems represented by the dipeptide permease Dpp, the tripeptide permease TppB, and the oligopeptide permease Opp [1][2][3]. Despite the fact that these proteins seem to discriminate according to the backbone length of peptide substrates, they do show some overlapping specificity. Although their prime physiological role is in the uptake of peptide-bound amino acids as an economic process to provide energy substrates and building blocks for cellular metabolism, peptide uptake seems also to be involved in signaling processes and metabolic adaptation [4]. Whereas Opp and Dpp represent ATP-binding cassette transporters with periplasmatic binding proteins [5][6][7], TppB belongs to the family of proton-dependent peptide symporters that utilize the proton gradient as driving force and lack cognate binding proteins [8]. After identification of the The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H + -dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.Abbreviations AMCA, b-Ala-Lys-N

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Section: Transport Modesupporting

confidence: 67%

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton‐dependent peptide transporters of Escherichia coli

et al. 2008

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“…The method is also particularly appealing due to the fact that the tethers are DNA based, suggesting compatibility with site-selective and sequence specific formation of arrays of planar lipid bilayers. This concept has so far only been realized for tethered lipid vesicles (Svedhem et al, 2003;Yoshina-Ishii and Boxer, 2003), in which case charge translocation measurements are far from straight forward (Kelety et al, 2006). Although such measurements require a sufficient solvent reservoir underneath the membrane, they are not expected to depend critically on the actual orientation of the DNA-tethers.…”

Section: Discussionmentioning

confidence: 99%

Self-assembly formation of multiple DNA-tethered lipid bilayers

Tabaei

Jönsson

Brändén

et al. 2009

Journal of Structural Biology

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“…In this respect, the effects of various inhibitors showing a variable affinity for SERCA were investigated using the SSM technique 22–24. It is worth mentioning that the SSM sensor combined with robotized instrumentation is expected to become an attractive platform technology for drug screening and development 25. 26…”

Section: Normalized Charges (Qn) Following 100 μM Atp Concentration Jmentioning

confidence: 99%

Computational Library Design for Increasing Haloalkane Dehalogenase Stability

et al. 2014

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We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app , 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

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Transporter Assays Using Solid Supported Membranes: A Novel Screening Platform for Drug Discovery

Cited by 43 publications

References 25 publications

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton‐dependent peptide transporters of Escherichia coli

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton‐dependent peptide transporters of Escherichia coli

Self-assembly formation of multiple DNA-tethered lipid bilayers

Computational Library Design for Increasing Haloalkane Dehalogenase Stability

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