This paper presented the dataset of correction parameters used in the determination of the energy transfer efficiencies from the spectrum-based fluorescence resonance energy transfer (FRET) measurement in a trimeric membrane protein AcrB. The cyan fluorescent protein (CFP) and yellow fluorescent protein (YPet) were used as the donor and acceptor, respectively. Two AcrB fusion proteins were constructed, AcrB-CFP and AcrB-YPet. The proteins were co-expressed in Escherichia coli cells, and energy transfer efficiency were determined in live cells. To obtain reliable energy transfer data, a complete set of correction parameters need to be first determined to accommodate for factors such as background fluorescence and spectra overlap. This paper described the methodology and determination of the correction factors, which are useful data and reference points for researchers working on fluorescence measurement of membrane protein complexes in live bacteria cells. Further interpretation and discussion of these data can be found in “Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter” (Wang et al., in press)

Cai, Yuguang

Wei, Yinan

Wilkop, Thomas E.

English

University of Kentucky

University of KentuckyUKnowledgeChemistry Faculty Publications Chemistry12-2018Data on Spectrum-Based Fluorescence ResonanceEnergy Transfer Measurement of E. coli MultidrugTransporter AcrBYuguang CaiUniversity of Kentucky, yuguang.cai@uky.eduThomas E. WilkopUniversity of Kentucky, Thomas.Wilkop@uky.eduYinan WeiUniversity of Kentucky, yinan.wei@uky.eduRight click to open a feedback form in a new tab to let us know how this document benefits you.Follow this and additional works at: https://uknowledge.uky.edu/chemistry_facpubPart of the Biochemistry Commons, Biophysics Commons, Cell Biology Commons, and theChemistry CommonsThis Article is brought to you for free and open access by the Chemistry at UKnowledge. It has been accepted for inclusion in Chemistry FacultyPublications by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu.Repository CitationCai, Yuguang; Wilkop, Thomas E.; and Wei, Yinan, "Data on Spectrum-Based Fluorescence Resonance Energy Transfer Measurementof E. coli Multidrug Transporter AcrB" (2018). Chemistry Faculty Publications. 158.https://uknowledge.uky.edu/chemistry_facpub/158Data on Spectrum-Based Fluorescence Resonance Energy Transfer Measurement of E. coli Multidrug TransporterAcrBNotes/Citation InformationPublished in Data in Brief, v. 21, p. 1649-1653.© 2018 The Author(s). Published by Elsevier Inc.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Digital Object Identifier (DOI)https://doi.org/10.1016/j.dib.2018.10.155This article is available at UKnowledge: https://uknowledge.uky.edu/chemistry_facpub/158Data ArticleData on spectrum-based fluorescence resonanceenergy transfer measurement of E. coli multidrugtransporter AcrBYuguang Cai a,n, Thomas Wilkop b, Yinan Wei a,na Department of Chemistry, University of Kentucky, Lexington, KY 40506, United Statesb Light Microscopy Core, University of Kentucky, Lexington, KY 40536, United Statesa r t i c l e i n f oArticle history:Received 15 October 2018Received in revised form25 October 2018Accepted 30 October 2018a b s t r a c tThis paper presented the dataset of correction parameters used inthe determination of the energy transfer efficiencies from thespectrum-based fluorescence resonance energy transfer (FRET)measurement in a trimeric membrane protein AcrB. The cyanfluorescent protein (CFP) and yellow fluorescent protein (YPet)were used as the donor and acceptor, respectively. Two AcrB fusionproteins were constructed, AcrB-CFP and AcrB-YPet. The proteinswere co-expressed in Escherichia coli cells, and energy transferefficiency were determined in live cells. To obtain reliable energytransfer data, a complete set of correction parameters need to befirst determined to accommodate for factors such as backgroundfluorescence and spectra overlap. This paper described the meth-odology and determination of the correction factors, which areuseful data and reference points for researchers working onfluorescence measurement of membrane protein complexes in livebacteria cells. Further interpretation and discussion of these datacan be found in “Comparison of in vitro and in vivo oligomericstates of a wild type and mutant trimeric inner membrane mul-tidrug transporter” (Wang et al., in press).& 2018 The Authors. Published by Elsevier Inc. This is an openaccess article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).Contents lists available at ScienceDirectjournal homepage: www.elsevier.com/locate/dibData in Briefhttps://doi.org/10.1016/j.dib.2018.10.1552352-3409/& 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).DOI of original article: https://doi.org/10.1016/j.bbrep.2018.10.006n Corresponding authors.E-mail addresses: Yuguang.cai@uky.edu (Y. Cai), yinan.wei@uky.edu (Y. Wei).Data in Brief 21 (2018) 1649–1653Specifications tableSubject area Biochemistry and biophysicsMore specific subject area Membrane protein fluorescence spectroscopyType of data Graph, figureHow data was acquired Zeiss LSM 880 with Airyscan laser scanning confocal microscopeData format AnalyzedExperimental factors E. coli cells transformed with the indicated plasmid was cultured to thelog phase and collected through centrifugation. The cell pellet wasresuspended in phosphate buffer and dropped to a 1% agar disk sup-ported on a microscopy cover slip. Cells were imaged directly withoutfurther treatment.Experimental features Protein of interest was tagged using a fluorescence protein domain, CFPor YPet. Energy transfer efficiency was measured using the spectrum-based fluorescence resonance energy transfer techniqueData source location Lexington, KY, 40513, UAData accessibility All data are presented in this article.Related research article Z. Wang, W. Lu, P. Rajapaksha, T. Wilkop, Y. Cai, Y. Wei Comparisonof in vitro and in vivo oligomeric states of a wild type and mutanttrimeric inner membrane multidrug transporter, in press [1]Value of the data The correction parameters determined and method described for the spectrum-based FRET can beused after adaptation by researcher conducting similar measurements. The correction method presented here eliminated the need of whole spectrum integration. The data can be used to reveal protein oligomeric state in live cell membranes.1. DataIn this article, we present data and related method to obtain fluorescence energy transfer effi-ciency of a trimeric membrane protein AcrB in live bacteria cell using a spectrum-based FRET method.Such data are useful in the determination of protein-protein interactions, especially for membraneintegrated proteins. Specifically, we first tagged AcrB with fluorescent proteins to construct AcrB-CFPand AcrB-YPet, and then we measured the FRET efficiency between CFP and YPet when the two fusionproteins were co-expressed in Escherichia coli cells. To determine the FRET efficiency, we firstdeveloped methodologies and determined a set of correction parameters.The fluorescent-labeled proteins are located in the E. coli membrane. In order to determine theemission spectra of the proteins, we performed an emission scan of the sample under excitation withthe 458 nm laser and segmented the E. coli membrane in the microscope image for analysis. Forrobustness sake, and to maximize the signal to noise ratio in the measurement, the segmentedregions of 20 cells were used to chart the representative spectral profile of the CFP and YPet, Fig. 1.The spectra shown in Fig. 1 were used to determine correction factors. The maximum emission peaksfor CFP and YPet are lying at 478 and 528 nm, respectively. CFP has significant emission near the YPetpeak, and hence potential spectral bleed through has to be corrected for in the FRET efficiency calculationwhen the 458 nm laser is used. The CFP bleed through coefficient (CFPBT ) is given by:CFPBT ¼R 533 nm523 nm I458CFP λ dλR 483 nm473 nm I458CFP λ dλð1Þwhere I458CFP λ is the CFP emission for excitation with the 458 nm laser (Fig. 1a). From the spectrumin Fig. 1a, CFPBT was determined as 0.556.Y. Cai et al. / Data in Brief 21 (2018) 1649–16531650The YPet spectra in Fig. 1b are the fluorescence emission spectrum of the YPet only sample excitedby 458 nm laser at 4% of its output power and by 514 nm laser at 1% of output power. The powersetting of the lasers was the same for all the subsequent FRET characterizations. The YPet cross-excitation coefficient (YPetCE) is given by:YPetCE ¼R 533 nm523 nm I458YPet λ dλR 533 nm523 nm I514YPet λ dλð2Þwhere I458YFP λ is the YPet emission spectrum excited at 458 nm (the blue line in Fig. 1b), I514YFP λ isthe YPet emission spectrum excited at 514 nm (the dark yellow line in Fig. 1b). From the two spectrain Fig. 1b, YPetCE was determined as 0.046.FRET determination E. coli cells expressing both AcrB-CFP and AcrB-YPet, or AcrBP223G-CFP andAcrBP223G-YPet were imaged in the channel mode. In channel 1 (C), the 458 nm laser at 4% power wasused as the excitation source, with the fluorescence signal between 473 and 483 nm was recorded, inChannel 2 (F), the fluorescence signal between 523 and 533 nm was recorded and in channel 3 (Y),using the 514 nm laser at 1% power the fluorescence between 523 and 533 nm was recorded. For allchannels the Airyscan detector was used with a gain setting of 800.The fluorescence signal (IF) recorded in Channel 2 (F) contains three components:IF ¼ IFRETþ IBTþ ICE ð3ÞThe first component (IFRET) is the true FRET signal. The second component (IBT) is the CFP fluor-escence signal that bleeds into the 523–528 nm band. IBT is determined by the CFP fluorescenceintensity ICFP multiplied by the CFP bleed through coefficient (CFPBT). The third component is the YPetcross excitation (ICE). ICE is calculated by multiplying the YPet fluorescence intensity at 514 nm (IYPet)with the YPet cross excitation coefficient (YPetCE).IF can hence be re-write as:IF ¼ IFRETþ ICFP  CFPBTþ IYFP  YPetCE ð4ÞThis can be solved for the true FRET signal:IFRET ¼ IF ICFP  CFPBT IYFP  YPetCE ð5ÞFig. 1. Representative emission spectra of AcrB-CFP (only acceptor labeled sample) (a) and AcrB-YPet (only donor labeledsample) (b). a) The blue and yellow bands indicate the spectral windows through the bandpass filter over which the fluor-escence signals were recorded by the multichannel Airyscan detector. The intensities in these windows were used in the FRETmeasurements. The multichannel detector recorded the fluorescence signal between 473–483 nm (Channel 1, “C”) band, andthe 523–533 nm (Channel 2 “F”) band using a 458 nm laser as the excitation source for imaging AraB-CFP. The ratio of thesignals is used to calculate CFPBT. b) The YPet spectra were recorded under excitation with the 458 nm laser, blue line and a514 nm laser, yellow line. The yellow region, of 523–533 nm (Channel 3, “Y”), indicates the transmission window for theAiryscan detector, when using either 514 or 458 nm laser line excitations. The respective areas under the curves are used tocalculate YPetCE.Y. Cai et al. / Data in Brief 21 (2018) 1649–1653 1651From the pure YPet emission spectrum IYPet(λ) plotted in Fig. 1b, the ratio between the overallemission intensity (IoverallFRET ) and the emission intensity between 523 and 533 nm (IFRET) was deter-mined to be:RYPet ¼R 593 nm463 nm IYFP λ dλR 533 nm523 nm IYFP λ dλ¼ 2:10 ð6ÞSimilarly, from the pure CFP emission spectrum, the ratio of overall CFP emission intensity (IoverallCFP )and the emission intensity recorded between 473 and 483 nm (ICFP) was determined to be:RCFP ¼R 593 nm463 nm ICFP λ dλR 483 nm473 nm ICFP λ dλ¼ 4:63 ð7ÞThe integration of spectral intensities over the range of 473–483 nm, or 523–533 nm, were used tocalculate the emission intensity of the entire spectra for CFP and YPet, respectively. [2].E¼ IAλDQD=QAIDλDþ IAλDQD=QA¼IFRETRYPetQCFPQYPetICFPRCFPþ IFRETRYPet QCFPQYPetð8ÞThis yielded values for QCFP of 0.40 and QYPet of 0.77 [3,4].Populating the expression for E with the various correction factors thus yields.E¼ 1:09ðIF0:556ICFP0:046IYPetÞ4:63ICFPþ1:09ðIF0:556ICFP0:046IYPetÞð9ÞWith, ICFP, IF, and IYPet being the intensity values experimentally obtained for channel 1 (C), Channel2 (F), and Channel 3 (Y), respectively.2. Experimental design, materials, and methodsA Zeiss LSM 880 confocal microscope with Airyscan was used to image E. coli cells deposited on agel pad, adopting the immobilization strategy outline in detail by Bottomley et al. [5]. The microscopecan be operated in Airyscan mode, in which a 32 element detector is used to image the Airy disc, inthis mode bandpass transmission filter are placed in the beam path, or it can be operated with photomultipliers that are picking up the fluorophore emissions over a user defined wavelength range orspectral scan. During our experiments both complementary modes were used. For standard imagingof the AcrB-CFP and YPet, the 458 nm and 514 nm laser line (at 1% power), respectively, were used forexcitation, with the fluorescence emission being collected between 473–483 nm (CFP) and 523–533nm (YPet), resulting in clear images of E. coli cells. AcrB is a membrane protein, the observed fluor-escence signal was characteristically for a membrane protein, concentrated in an envelope -likeprofile in the E. coli cells.AcknowledgmentsWe thank the UK Light Microscopy Core for the usage of the Zeiss LSM 880 microscope andtechnical support. We thank Dr. Chris Richards for valuable discussions. This study is supported byNSF Grant CHE-1709381 (YW).Transparency document. Supplementary materialTransparency document associated with this article can be found in the online version at https://doi.org/10.1016/j.dib.2018.10.155.Y. Cai et al. / Data in Brief 21 (2018) 1649–16531652References[1] Z. Wang, W. Lu, P. Rajapaksha, T. Wilkop, Y. Cai, Y. Wei, Comparison of in vitro and in vivo oligomeric states of a wild typeand mutant trimeric inner membrane multidrug transporter, Biochem. Biophys. Reports 16 (2018) 122–129.[2] D. Megias, R. Marrero, B.M. Del Peso, M.A. Garcia, J.J. Bravo-Cordero, A. Garcia-Grande, A. Santos, M.C. Montoya, Novellambda fret spectral confocal microscopy imaging method, Microsc. Res. Tech. 72 (2009) 1–11.[3] G. Patterson, R.N. Day, D. Piston, Fluorescent protein spectra, J. Cell Sci. 114 (2001) 837–838.[4] W. Nguyen, P.S. Daugherty, Evolutionary optimization of fluorescent proteins for intracellular FRET, Nat. Biotechnol.23 (2005) 355–360.[5] L. Bottomley, L. Turnbull, C.B. Whitchurch, E.J. Harry, Immobilization techniques of bacteria for live super-resolutionimaging using structured illumination microscopy, Methods Mol. Biol. 1535 (2017) 197–209.Y. Cai et al. / Data in Brief 21 (2018) 1649–1653 1653

Data on Spectrum-Based Fluorescence Resonance Energy Transfer Measurement of \u3cem\u3eE. coli\u3c/em\u3e Multidrug Transporter AcrB

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Data on Spectrum-Based Fluorescence Resonance Energy Transfer Measurement of \u3cem\u3eE. coli\u3c/em\u3e Multidrug Transporter AcrB

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