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

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 becaus...

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Autores principales: Qazi, S. Junaid S., Chew, Raymond, Bay, Denice C., Turner, Raymond J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668558/
https://www.ncbi.nlm.nih.gov/pubmed/29124131
http://dx.doi.org/10.1016/j.bbrep.2015.03.007
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author Qazi, S. Junaid S.
Chew, Raymond
Bay, Denice C.
Turner, Raymond J.
author_facet Qazi, S. Junaid S.
Chew, Raymond
Bay, Denice C.
Turner, Raymond J.
author_sort Qazi, S. Junaid S.
collection PubMed
description 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 (His(6)) 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 (K(d)) and maximum specific one-site binding (B(max)) 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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spelling pubmed-56685582017-11-09 Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE Qazi, S. Junaid S. Chew, Raymond Bay, Denice C. Turner, Raymond J. Biochem Biophys Rep Research Article 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 (His(6)) 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 (K(d)) and maximum specific one-site binding (B(max)) 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. Elsevier 2015-03-26 /pmc/articles/PMC5668558/ /pubmed/29124131 http://dx.doi.org/10.1016/j.bbrep.2015.03.007 Text en © 2015 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research Article
Qazi, S. Junaid S.
Chew, Raymond
Bay, Denice C.
Turner, Raymond J.
Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE
title Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE
title_full Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE
title_fullStr Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE
title_full_unstemmed Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE
title_short Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE
title_sort structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, emre
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668558/
https://www.ncbi.nlm.nih.gov/pubmed/29124131
http://dx.doi.org/10.1016/j.bbrep.2015.03.007
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