Visualizing a multidrug resistance protein, EmrE, with major bacterial lipids using Brewster angle microscopy

Nathoo, Safia; Litzenberger, Jennifer K.; Bay, Denice C.; Turner, Raymond J.; Prenner, Elmar J.

doi:10.1016/j.chemphyslip.2013.01.007

Cited by 19 publications

(13 citation statements)

References 71 publications

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“…Taking this into consideration, the presence of two peaks for T-EmrE suggested that it consist of primarily two different multimeric states whereas the third peak in UT-EmrE may represent a higher multimeric form. Large aggregations of EmrE have been observed previously in lipid domain experiments where lipid type and lateral membrane pressure may play a role [34] , [35] .…”

Section: Discussionsupporting

confidence: 61%

Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE

Qazi

Chew

Bay

et al. 2015

Biochemistry and Biophysics Reports

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EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His6) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-β-d-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants (Kd) and maximum specific one-site binding (Bmax) values for particular QCCs. In vitro analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.

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Section: Discussionsupporting

confidence: 61%

Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE

Qazi

Chew

Bay

et al. 2015

Biochemistry and Biophysics Reports

Self Cite

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“…Upon compression, the surface pressure ( Π ) was measured as a function of the area of water surface available to each lipid molecule ( A ). The surface pressure–area ( Π–A ) isotherms that were obtained for each sample (Figure S1 in the Supporting Information) were in good agreement with previously reported data for DMPG and DMPC monolayers 38–40. The thioether–cholesterol ligand 1 alone also forms monolayers.…”

Section: Resultssupporting

confidence: 89%

“…The surface pressure-area (P-A) isotherms that were obtained for each sample ( Figure S1 in the Supporting Information) were in good agreement with previously reported data for DMPG and DMPC monolayers. [38][39][40] The thioether-cholesterol ligand 1 alone also forms monolayers. The ligand monolayer was formed, but it was not very stable.…”

Section: Langmiur-blodgett Monolayer Experimentsmentioning

confidence: 99%

Binding of a Ruthenium Complex to a Thioether Ligand Embedded in a Negatively Charged Lipid Bilayer: A Two‐Step Mechanism

Bahreman

Rabe

Kros

et al. 2014

Chemistry A European J

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The interaction between the ruthenium polypyridyl complex [Ru(terpy)(dcbpy)(H2O)](2+) (terpy = 2,2';6',2"-terpyridine, dcbpy = 6,6'-dichloro-2,2'-bipyridine) and phospholipid membranes containing either thioether ligands or cholesterol were investigated using UV-visible spectroscopy, Langmuir-Blodgett monolayer surface pressure measurements, and isothermal titration calorimety (ITC). When embedded in a membrane, the thioether ligand coordinated to the dicationic metal complex only when the phospholipids of the membrane were negatively charged, that is, in the presence of attractive electrostatic interaction. In such a case coordination is much faster than in homogeneous conditions. A two-step model for the coordination of the metal complex to the membrane-embedded sulfur ligand is proposed, in which adsorption of the complex to the negative surface of the monolayers or bilayers occurs within minutes, whereas formation of the coordination bond between the surface-bound metal complex and ligand takes hours. Finally, adsorption of the aqua complex to the membrane is driven by entropy. It does not involve insertion of the metal complex into the hydrophobic lipid layer, but rather simple electrostatic adsorption at the water-bilayer interface.

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“…Further analyses of the intact dimer have shown stoichiometric lipid binding and posttranslational modifications (PTMs) by MS (Barrera et al, 2009 ) and identification of a specific residue involved in the substrate binding site of tetraphenylphosphonium by magic angle spinning-NMR (Ong et al, 2013 ). The interaction between annular lipids and EmrE has also been tested using Brewster angle microscopy (Nathoo et al, 2013 ), which showed longer unsaturated chains of cardiolipin (CL) forming the most stable monolayer in the presence of EmrE. Once a MP-ligand complex has been identified, especially for those lipids already co-purified from a cellular expression system, complementary results for their effect over soluble ligand binding can be provided by AFM, SPR, ITC, and FRET.…”

Section: Biophysical Methods To Analyze Membrane Protein-lipid Bindinmentioning

confidence: 99%

Combining Mass Spectrometry and X-Ray Crystallography for Analyzing Native-Like Membrane Protein Lipid Complexes

2017

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Membrane proteins represent a challenging family of macromolecules, particularly related to the methodology aimed at characterizing their three-dimensional structure. This is mostly due to their amphipathic nature as well as requirements of ligand bindings to stabilize or control their function. Recently, Mass Spectrometry (MS) has become an important tool to identify the overall stoichiometry of native-like membrane proteins complexed to ligand bindings as well as to provide insights into the transport mechanism across the membrane, with complementary information coming from X-ray crystallography. This perspective article emphasizes MS findings coupled with X-ray crystallography in several membrane protein lipid complexes, in particular transporters, ion channels and molecular machines, with an overview of techniques that allows a more thorough structural interpretation of the results, which can help us to unravel hidden mysteries on the membrane protein function.

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Visualizing a multidrug resistance protein, EmrE, with major bacterial lipids using Brewster angle microscopy

Cited by 19 publications

References 71 publications

Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE

Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE

Binding of a Ruthenium Complex to a Thioether Ligand Embedded in a Negatively Charged Lipid Bilayer: A Two‐Step Mechanism

Combining Mass Spectrometry and X-Ray Crystallography for Analyzing Native-Like Membrane Protein Lipid Complexes

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