{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T21:13:41Z","timestamp":1788470021827,"version":"build-2803163510"},"reference-count":51,"publisher":"American Chemical Society (ACS)","issue":"23","license":[{"start":{"date-parts":[[2015,5,22]],"date-time":"2015-05-22T00:00:00Z","timestamp":1432252800000},"content-version":"vor","delay-in-days":365,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["MCB 1158036"],"award-info":[{"award-number":["MCB 1158036"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001677","name":"National institute of Health and Medical Research","doi-asserted-by":"publisher","award":["1R21AI103717"],"award-info":[{"award-number":["1R21AI103717"]}],"id":[{"id":"10.13039\/501100001677","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2014,6,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The majority of membrane proteins function as oligomers. However, it remains largely unclear how the oligomer stability of protein complexes correlates with their function. Understanding the relationship between oligomer stability and activity is essential to protein research and to virtually all cellular processes that depend on the function of protein complexes. Proteins make lasting or transient interactions as they perform their functions. Obligate oligomeric proteins exist and function exclusively at a specific oligomeric state. Although oligomerization is clearly critical for such proteins to function, a direct correlation between oligomer affinity and biological activity has not yet been reported. Here, we used an obligate trimeric membrane transporter protein, AcrB, as a model to investigate the correlation between its relative trimer affinity and efflux activity. AcrB is a component of the major multidrug efflux system in Escherichia coli. We created six AcrB constructs with mutations at the transmembrane intersubunit interface, and we determined their activities using both a drug susceptibility assay and an ethidium bromide accumulation assay. The relative trimer affinities of these mutants in detergent micelles were obtained using blue native polyacrylamide gel electrophoresis. A correlation between the relative trimer affinity and substrate efflux activity was observed, in which a threshold trimer stability was required to maintain efflux activity. The trimer affinity of the wild-type protein was approximately 3 kcal\/mol more stable than the threshold value. Once the threshold was reached, an additional increase of stability in the range observed had no observable effect on protein activity.<\/jats:p>","DOI":"10.1021\/bi5000838","type":"journal-article","created":{"date-parts":[[2014,5,22]],"date-time":"2014-05-22T11:36:42Z","timestamp":1400758602000},"page":"3738-3746","source":"Crossref","is-referenced-by-count":10,"title":["Correlation between AcrB Trimer Association Affinity\nand Efflux Activity"],"prefix":"10.1021","volume":"53","author":[{"given":"Cui","family":"Ye","sequence":"first","affiliation":[{"name":"University of Kentucky , , , ,","place":["Lexington, Kentucky, United States, 40506"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaoshuai","family":"Wang","sequence":"additional","affiliation":[{"name":"University of Kentucky , , , ,","place":["Lexington, Kentucky, United States, 40506"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Lu","sequence":"additional","affiliation":[{"name":"University of Kentucky , , , ,","place":["Lexington, Kentucky, United States, 40506"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meng","family":"Zhong","sequence":"additional","affiliation":[{"name":"University of Kentucky , , , ,","place":["Lexington, Kentucky, United States, 40506"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qian","family":"Chai","sequence":"additional","affiliation":[{"name":"University of Kentucky , , , ,","place":["Lexington, Kentucky, United States, 40506"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yinan","family":"Wei","sequence":"additional","affiliation":[{"name":"University of Kentucky , , , ,","place":["Lexington, Kentucky, United States, 40506"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2014,5,22]]},"reference":[{"key":"2026090316180814500_cit1","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1038\/328834a0","article-title":"The accessible surface\narea and stability of oligomeric proteins","volume":"328","author":"Miller","year":"1987","journal-title":"Nature"},{"key":"2026090316180814500_cit2","doi-asserted-by":"crossref","first-page":"14243","DOI":"10.1073\/pnas.93.25.14243","article-title":"Symmetry,\nstability, and dynamics of multidomain and\nmulticomponent protein systems","volume":"93","author":"Blundell","year":"1996","journal-title":"Proc. Natl.\nAcad. Sci. U.S.A."},{"key":"2026090316180814500_cit3","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1073\/pnas.92.2.452","article-title":"A relationship between\nprotein stability and protein function","volume":"92","author":"Shoichet","year":"1995","journal-title":"Proc.\nNatl. Acad. Sci. U.S.A."},{"key":"2026090316180814500_cit4","volume-title":"Structure\nand Mechanism in Protein Science","author":"Fersht","year":"1999"},{"key":"2026090316180814500_cit5","doi-asserted-by":"crossref","first-page":"5869","DOI":"10.1073\/pnas.0510098103","article-title":"Protein stability\npromotes evolvability","volume":"103","author":"Bloom","year":"2006","journal-title":"Proc. Natl. Acad. Sci.\nU.S.A."},{"key":"2026090316180814500_cit6","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1016\/S1095-6433(00)00359-7","article-title":"Protein\nfunction at thermal extremes: balancing stability and flexibility","volume":"129","author":"Fields","year":"2001","journal-title":"Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol."},{"key":"2026090316180814500_cit7","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.1515\/hsz-2012-0231","article-title":"Oligomerization\nof polytopic \u03b1-helical membraneproteins:\ncauses and consequences","volume":"393","author":"Cymer","year":"2012","journal-title":"Biol. Chem."},{"key":"2026090316180814500_cit8","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/S0076-6879(08)04208-0","article-title":"Methods for measuring\nthe thermodynamic stability of membrane proteins","volume":"455","author":"Hong","year":"2009","journal-title":"Methods Enzymol."},{"key":"2026090316180814500_cit9","doi-asserted-by":"crossref","first-page":"691","DOI":"10.1111\/j.1751-7915.2010.00189.x","article-title":"Characterization\nof the RND family of multidrug efflux pumps: in silico to in vivo confirmation of four functionally distinct subgroups","volume":"3","author":"Godoy","year":"2010","journal-title":"Microbiol. Biotechnol."},{"key":"2026090316180814500_cit10","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1016\/j.mib.2009.07.003","article-title":"Structure, function and inhibition\nof RND efflux pumps in Gram-negative bacteria: an update","volume":"12","author":"Blair","year":"2009","journal-title":"Curr. Opin. Microbiol."},{"key":"2026090316180814500_cit11","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1016\/j.bbapap.2008.10.004","article-title":"Mechanisms of RND multidrug efflux pumps","volume":"1794","author":"Nikaido","year":"2009","journal-title":"Biochim. Biophys. Acta"},{"key":"2026090316180814500_cit12","doi-asserted-by":"crossref","first-page":"1729","DOI":"10.1128\/JB.01441-08","article-title":"Covalently linked trimer of the AcrB multidrug efflux\npump provides support for the functional rotating mechanism","volume":"191","author":"Takatsuka","year":"2009","journal-title":"J. Bacteriol."},{"key":"2026090316180814500_cit13","doi-asserted-by":"crossref","first-page":"7173","DOI":"10.1073\/pnas.0900693106","article-title":"The assembled\nstructure of a complete tripartite bacterial multidrug efflux pump","volume":"106","author":"Symmons","year":"2009","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316180814500_cit14","doi-asserted-by":"crossref","first-page":"32116","DOI":"10.1074\/jbc.M402230200","article-title":"AcrA, AcrB, and TolC of Escherichia coli form a stable intermembrane multidrug efflux complex","volume":"279","author":"Tikhonova","year":"2004","journal-title":"J. Biol. Chem."},{"key":"2026090316180814500_cit15","doi-asserted-by":"crossref","first-page":"11115","DOI":"10.1021\/bi050452u","article-title":"Direct interaction\nof multidrug efflux transporter AcrB and outer membrane channel TolC\ndetected via site-directed disulfide cross-linking","volume":"44","author":"Tamura","year":"2005","journal-title":"Biochemistry"},{"key":"2026090316180814500_cit16","doi-asserted-by":"crossref","first-page":"16416","DOI":"10.1073\/pnas.0906601106","article-title":"Kinetic\ncontrol of TolC recruitment by multidrug efflux complexes","volume":"106","author":"Tikhonova","year":"2009","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316180814500_cit17","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.1126\/science.1131542","article-title":"Structural asymmetry of AcrB trimer suggests a peristaltic\npump mechanism","volume":"313","author":"Seeger","year":"2006","journal-title":"Science"},{"key":"2026090316180814500_cit18","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1038\/nature05076","article-title":"Crystal structures of a multidrug\ntransporter reveal\na functionally rotating mechanism","volume":"443","author":"Murakami","year":"2006","journal-title":"Nature"},{"key":"2026090316180814500_cit19","doi-asserted-by":"crossref","first-page":"e28390","DOI":"10.1371\/journal.pone.0028390","article-title":"AcrB trimer\nstability and efflux activity, insight\nfrom mutagenesis studies","volume":"6","author":"Yu","year":"2011","journal-title":"PLoS One"},{"key":"2026090316180814500_cit20","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1080\/07391102.2012.703065","article-title":"Dissecting the function\nof a protruding loop in AcrB trimerization","volume":"31","author":"Fang","year":"2013","journal-title":"J. Biomol. Struct. Dyn."},{"key":"2026090316180814500_cit21","doi-asserted-by":"crossref","first-page":"863","DOI":"10.2174\/092986611796011446","article-title":"A reporter platform for the monitoring\nof in vivo conformational changes in AcrB","volume":"18","author":"Lu","year":"2011","journal-title":"Protein Pept. Lett."},{"key":"2026090316180814500_cit22","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.jmb.2012.06.036","article-title":"Assembling\nof AcrB trimer in cell membrane","volume":"423","author":"Lu","year":"2012","journal-title":"J. Mol. Biol."},{"key":"2026090316180814500_cit23","first-page":"36","article-title":"Image processing with ImageJ","volume":"11","author":"Abramoff","year":"2004","journal-title":"Biophoton. Int."},{"key":"2026090316180814500_cit24","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.jmb.2011.05.042","article-title":"Folding of AcrB subunit precedes\ntrimerization","volume":"411","author":"Lu","year":"2011","journal-title":"J. Mol. Biol."},{"key":"2026090316180814500_cit25","doi-asserted-by":"publisher","DOI":"10.1002\/pro.2471","article-title":"Unfolding study of a trimeric\nmembrane\nprotein AcrB","author":"Ye","year":"2014","journal-title":"Protein Sci."},{"key":"2026090316180814500_cit26","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1128\/AAC.47.1.27-33.2003","article-title":"Contributions\nof MexAB-OprM and an\nEmrE homolog to intrinsic resistance of Pseudomonas aeruginosa to\naminoglycosides and dyes","volume":"47","author":"Li","year":"2003","journal-title":"Antimicrob. Agents\nChemother."},{"key":"2026090316180814500_cit27","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1093\/bioinformatics\/btn584","article-title":"ProtorP: a\nprotein\u2013protein\ninteraction analysis server","volume":"25","author":"Reynolds","year":"2009","journal-title":"Bioinformatics"},{"key":"2026090316180814500_cit28","doi-asserted-by":"crossref","first-page":"6790","DOI":"10.1021\/bi400452v","article-title":"Role of a conserved residue R780\nin Escherichia coli multidrug transporter AcrB","volume":"52","author":"Yu","year":"2013","journal-title":"Biochemistry"},{"key":"2026090316180814500_cit29","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1016\/j.ijantimicag.2007.12.015","article-title":"Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide\nmethod","volume":"31","author":"Viveiros","year":"2008","journal-title":"Int. J. Antimicrob. Agents"},{"key":"2026090316180814500_cit30","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1186\/1754-1611-3-18","article-title":"Fluorometric determination of ethidium\nbromide efflux kinetics in Escherichia coli","volume":"3","author":"Paixao","year":"2009","journal-title":"J. Biol. Eng."},{"key":"2026090316180814500_cit31","doi-asserted-by":"crossref","first-page":"3770","DOI":"10.1128\/AAC.00620-10","article-title":"Optimized\nNile Red efflux assay of\nAcrAB-TolC multidrug efflux system shows competition between substrates","volume":"54","author":"Bohnert","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"2026090316180814500_cit32","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1016\/S0022-2836(02)00920-8","article-title":"Standardizing\nthe free energy change of transmembrane helix\u2013helix interactions","volume":"323","author":"Fleming","year":"2002","journal-title":"J. Mol. Biol."},{"key":"2026090316180814500_cit33","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.bbamem.2005.08.006","article-title":"Using micellar mole fractions to\nassess membrane protein stability in mixed micelles","volume":"1716","author":"Sehgal","year":"2005","journal-title":"Biochim. Biophys. Acta"},{"key":"2026090316180814500_cit34","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1038\/nprot.2006.62","article-title":"Blue native PAGE","volume":"1","author":"Wittig","year":"2006","journal-title":"Nat. Protoc."},{"key":"2026090316180814500_cit35","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1006\/jmbi.2002.5416","article-title":"Oligomeric\nstate of membrane transport proteins analyzed with blue native electrophoresis\nand analytical ultracentrifugation","volume":"317","author":"Heuberger","year":"2002","journal-title":"J. Mol.\nBiol."},{"key":"2026090316180814500_cit36","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1021\/bi034875c","article-title":"Estimation of helix\u2013helix\nassociation free energy from partial unfolding of bacterioopsin","volume":"43","author":"Nannepaga","year":"2004","journal-title":"Biochemistry"},{"key":"2026090316180814500_cit37","doi-asserted-by":"crossref","first-page":"522","DOI":"10.1016\/j.bbamem.2008.11.011","article-title":"Sensory rhodopsin II\/transducer complex formation in\ndetergent and in lipid bilayers studied with FRET","volume":"1788","author":"Kriegsmann","year":"2009","journal-title":"Biochim. Biophys. Acta"},{"key":"2026090316180814500_cit38","doi-asserted-by":"crossref","first-page":"3954","DOI":"10.1021\/bi981795d","article-title":"Depolymerization of phospholamban\nin the presence of calcium pump: a fluorescence energy transfer study","volume":"38","author":"Reddy","year":"1999","journal-title":"Biochemistry"},{"key":"2026090316180814500_cit39","doi-asserted-by":"crossref","first-page":"5539","DOI":"10.1021\/bi00184a024","article-title":"Glycophorin A helical transmembrane domains dimerize\nin phospholipid bilayers: a resonance energy transfer study","volume":"33","author":"Adair","year":"1994","journal-title":"Biochemistry"},{"key":"2026090316180814500_cit40","doi-asserted-by":"crossref","first-page":"4544","DOI":"10.1074\/jbc.M109.034512","article-title":"The structure of calreticulin C-terminal\ndomain is modulated by physiological variations of calcium concentration","volume":"285","author":"Villamil Giraldo","year":"2010","journal-title":"J. Biol. Chem."},{"key":"2026090316180814500_cit41","doi-asserted-by":"crossref","first-page":"13643","DOI":"10.1021\/jp2035289","article-title":"Multidonor\ndeep-UV FRET study of protein\u2013ligand\nbinding and its potential to obtain structure information","volume":"115","author":"Li","year":"2011","journal-title":"Phys. Chem. B"},{"key":"2026090316180814500_cit42","doi-asserted-by":"crossref","first-page":"14772","DOI":"10.1073\/pnas.2536751100","article-title":"Use of thiol-disulfide equilibria\nto measure the energetics of assembly of transmembrane helices in\nphospholipid bilayers","volume":"100","author":"Cristian","year":"2003","journal-title":"Proc. Natl. Acad. Sci.\nU.S.A."},{"key":"2026090316180814500_cit43","doi-asserted-by":"crossref","first-page":"19802","DOI":"10.1073\/pnas.1010348107","article-title":"Method to measure\nstrong protein-protein interactions in lipid bilayers using a steric\ntrap","volume":"107","author":"Hong","year":"2010","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316180814500_cit44","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1146\/annurev.bi.39.070170.000325","article-title":"Quaternary structure of proteins","volume":"39","author":"Klotz","year":"1970","journal-title":"Annu. Rev. Biochem."},{"key":"2026090316180814500_cit45","doi-asserted-by":"crossref","first-page":"5013","DOI":"10.1016\/j.bmc.2005.05.037","article-title":"Protein oligomerization:\nhow and why","volume":"13","author":"Ali","year":"2005","journal-title":"Biorg. Med. Chem."},{"key":"2026090316180814500_cit46","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1146\/annurev.biophys.29.1.105","article-title":"Structural symmetry\nand protein function","volume":"29","author":"Goodsell","year":"2000","journal-title":"Annu. Rev. Biophys. Biomol.\nStruct."},{"key":"2026090316180814500_cit47","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/0968-0004(91)90083-8","article-title":"Inside\na living cell","volume":"16","author":"Goodsell","year":"1991","journal-title":"Trends Biochem. Sci."},{"key":"2026090316180814500_cit48","doi-asserted-by":"crossref","first-page":"3486","DOI":"10.1093\/emboj\/cdg359","article-title":"Diversity of protein\u2013protein\ninteractions","volume":"22","author":"Nooren","year":"2003","journal-title":"EMBO J."},{"key":"2026090316180814500_cit49","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1006\/jmbi.1997.1236","article-title":"The effect of point\nmutations on\nthe free energy of transmembrane alpha-helix dimerization","volume":"272","author":"Fleming","year":"1997","journal-title":"J. Mol. Biol."},{"key":"2026090316180814500_cit50","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1016\/j.ab.2005.01.035","article-title":"FRET in liposomes: measurements of\nTM helix dimerization in the native bilayer environment","volume":"340","author":"You","year":"2005","journal-title":"Anal. Biochem."},{"key":"2026090316180814500_cit51","doi-asserted-by":"crossref","first-page":"3628","DOI":"10.1021\/ja910692u","article-title":"Measuring\nthe energetics of membrane protein dimerization in mammalian membranes","volume":"132","author":"Chen","year":"2010","journal-title":"J. Am. Chem. Soc."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/bi5000838","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/53\/23\/3738\/7891463\/bi5000838.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/53\/23\/3738\/7891463\/bi5000838.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T20:18:25Z","timestamp":1788466705000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/53\/23\/3738\/774324\/Correlation-between-AcrB-Trimer-Association"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,5,22]]},"references-count":51,"journal-issue":{"issue":"23","published-online":{"date-parts":[[2014,5,22]]},"published-print":{"date-parts":[[2014,6,17]]}},"URL":"https:\/\/doi.org\/10.1021\/bi5000838","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,5,22]]}}}