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PMID:20036636

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Citation

Bay, DC, Budiman, RA, Nieh, MP and Turner, RJ (2010) Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent. Biochim. Biophys. Acta 1798:526-35

Abstract

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane alpha-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (</=1muM) in SDS-EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS-EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (>/=100nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS-EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.

Links

PubMed Online version:10.1016/j.bbamem.2009.12.017

Keywords

Antiporters/chemistry; Antiporters/metabolism; Circular Dichroism; Detergents/pharmacology; Electrophoresis, Polyacrylamide Gel; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Models, Molecular; Multidrug Resistance-Associated Proteins/chemistry; Multidrug Resistance-Associated Proteins/metabolism; Neutron Diffraction; Onium Compounds/pharmacology; Organophosphorus Compounds/pharmacology; Protein Multimerization/drug effects; Protein Structure, Secondary; Scattering, Small Angle; Sodium Dodecyl Sulfate/pharmacology; Solubility/drug effects; Solutions; Spectrometry, Fluorescence

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