Abstract
EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. It confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by the transmembrane proton motive force. We have expressed hexahistidinyl (His6) – myc epitope tagged EmrE, extracted it from membrane preparations using the detergent n-dodecyl-β-D-maltopyranoside (DDM), and purified it using nickel-affinity chromatography. The size of the EmrE protein, in DDM environment, was then examined in the presence and absence of a range of structurally different QCC ligands that varied in their chemical structure, charge and shape. We used dynamic light scattering and showed that the size and oligomeric state distributions are dependent on the type of QCC. We also followed changes in the Trp fluorescence and determined apparent dissociation constants (Kd). Overall, our in vitro analyses of epitope tagged EmrE demonstrated subtle but significant differences in the size distributions with different QCC ligands bound.
Highlights
Bacterial antiseptic and antibiotic resistance present major challenges in controlling infection, in healthcare settings [1,2,3,4]
EmrE was examined in the presence of 19 different quaternary cationic compounds (QCCs) ligands
The QCCs were divided into four categories: sphere forming (TPA, Methyltriphenyl phosphonium bromide (MTP), TTP), polyaromatic (ACR, PRO, Crystal Violet (CV), Rhodamine 6G (RH), Pyronin Y (PY), HE, Ethidium bromide (EB)), acyl-chained (BZ, MC, CTC, CET, Cetylpyridinium bromide (CB), Stearyltrimethylammonium chloride (STAC), CTP), and poly-charged (DC, Methyl Viologen (MV))
Summary
Bacterial antiseptic and antibiotic resistance present major challenges in controlling infection, in healthcare settings [1,2,3,4]. EmrE is one of many transporters responsible for antiseptic drug resistance in bacteria, and catalyzes the efflux of quaternary cationic compounds (QCCs) [7,8] It comprises 4 transmembrane alpha helices, connected by short loops (as reviewed by [6]), and has been shown to exist as a monomer, dimer, trimer, tetramer or even higher ordered multimers/ complexes, but the minimal functional unit is considered to be a dimer [9,10,11,12,13]. A widely used purification method for EmrE involves the use of a hexahistidine (His6) tag at its C-terminus, which facilitates protein purification using Ni2+-affinity chromatography [14] This His tag is usually left on the protein in most structural analyses (as reviewed by [6]). Because of this extensive use of a tagged version of EmrE protein by other research groups and its use in most of the structural biology studies, it is very important to explore the ligand binding behavior of this version of EmrE protein with respect to the wide range of QCC ligand substrates
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
