Spectroscopic analysis of small multidrug resistance protein EmrE in the presence of various quaternary cation compounds.
Escherichia coli EmrE protein is the archetypical member of the small multidrug resistance protein family in bacteria and confers host resistance to a wide assortment of toxic quaternary cation compounds by secondary active efflux. This protein can form a variety of multimers under various membrane mimetic conditions, and the consensus of most biochemical and biophysical studies indicate that the active form is a dimer. The purpose of this study is to characterize the conformation of organically extracted detergent solubilized EmrE protein known to predominate as monomer yet demonstrates ligand binding ability. Active site EmrE-E14 replacements were also examined as functionally inactive controls for this study. EmrE was solubilized in detergents, sodium dodecyl sulfate (SDS) and dodecyl maltoside (DDM), and protein conformation was examined in the presence of four known quaternary cation compound (QCC) substrates, tetraphenyl phosphonium (TPP), methyl viologen, cetylpyridinium, and ethidium. SDS-Tricine PAGE analysis of both detergent solubilized proteins revealed that DDM-EmrE preparations enhanced the formation of dimer (and in some cases trimer) forms in the presence of all four QCC above 25 QCC:1 EmrE molar ratios. Examination of EmrE and its active site variant tertiary structures in DDM by circular dichroism spectropolarimetry, intrinsic Trp fluorescence quenching and second order derivative ultraviolet absorbance revealed that the variant fails to bind TPP but interacts with all other compounds. The results of this study show that monomeric detergent solubilized EmrE is capable of forming multimeric complexes that are enhanced by chemically diverse QCCs.
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4.
Denice C
DC
Bay
Raymond J
RJ
Turner
D017920:Antiporters / Q000737:chemistry*; D002412:Cations / Q000737:chemistry; D002594:Cetylpyridinium / Q000737:chemistry*; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D004996:Ethidium / Q000737:chemistry*; D005960:Glucosides / Q000737:chemistry; D009861:Onium Compounds / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010269:Paraquat / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D017920:Antiporters; D002412:Cations; D029968:Escherichia coli Proteins; D005960:Glucosides; D009861:Onium Compounds; D009943:Organophosphorus Compounds; C075436:EmrE protein, E coli; D012967:Sodium Dodecyl Sulfate; C040358:dodecyl maltoside; D002594:Cetylpyridinium; D004996:Ethidium; D010269:Paraquat; C013289:tetraphenylphosphonium