Abstract

Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam. For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps, we measured FRET from cyan (CFP) to yellow fluorescent protein (YFP) by spectral analysis and fluorescence lifetime imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response to pharmaceutical agents, e.g., Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples.

Highlights

  • Measurement of Förster resonance energy transfer (FRET) [1] between a donor and an acceptor molecule or between different chromophoric groups of a larger molecule, e.g., a protein, has become a valuable tool for probing either molecular interactions or conformational changes of a molecule in the nanometer range

  • Intermolecular FRET between epidermal growth factor receptor (EGFR)‐CFP and Grb2‐yellow fluorescent protein (YFP) In Figure, two total internal reflection (TIR) fluorescence images are depicted for HeLa cervix carcinoma cells stably transfectIend FwigituhreG,rtbw2-oYTFPIR, afnludotrreasncseinencetlyimtraagnessfeacrteeddwepitihcteEdGFfoRr-CHFePLawictehrovuixt fcuarrtchienromstiamcuellalstiosnta.bly AftetrraonpstfieccatledexwciittahtiGonrbo2f‐YCFFPP, aant d42t0ra–n44si0enntmly, twraenrsefeccotreddedwibtlhuEeGflFuRo‐rCesFcPenwciethinoutht efusrptheectrrsatlimraunlgaetion. 450–A4f9t0ernmop(tFicigalureexc2i,tlaetfito)nanodf gCrFePena-yt e4l2lo0w–4fl40uonrmes,cwenecereactoλrd≥e5d1b0lnume f(lFuiogruersec2e,nrcieghint).tThehesspeescptreacltrraalnge ima4g5e0s–r4e9s0ulntmfro(mFigthuerebr2i,glhetfte)mainsdsiognreoefnd‐yoenlolorw(CfFluPo)raensdceanccceepattoλr (≥YF51P0) mnmole(cFuigleusredi2st,rribiguhtte)d

  • We conclude that measurements of FRET between fluorescent proteins under TIR illumination are appropriate to detect molecular interactions or changes of molecular conformation due to pharmaceutical agents in close proximity to the plasma membrane

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Summary

Introduction

Measurement of Förster resonance energy transfer (FRET) [1] between a donor and an acceptor molecule (intermolecular FRET) or between different chromophoric groups of a larger molecule, e.g., a protein (intramolecular FRET), has become a valuable tool for probing either molecular interactions or conformational changes of a molecule in the nanometer range. The method is based on optical excitation of a so-called donor molecule and interaction of optical transition dipoles with an acceptor molecule, which is able to fluoresce. FRET measurements of living cells have been reported for about 30 years [6,7], but recently their plasma membranes have bInet.eJn. Maosls. Iτnigs tthoe reciprocal of all rates of deactivation of an excited molecular state, and if a lifetime τ0 is determined for a reference system without FRET, the difference between Haderdeibtiyo,nFtRoEiTntfernosmityCmFPeatsouYreFmPeinstesxoafmdionneodr, aanndd acoccmeppatorerdflutoortehsecewnhcoel,emceeallseuxrpemereimntesnotsf trheepoflruteodreisncethneceliltiefreatitmureeτ[2o4f,2t5h,e26d]o, nToIRr mexopleecriumleen(CtsFwP)eirne tmhoerneansoelseecctoivned rfoanr gdeeatreecteioxnpecotfedmtoemgbivreanreeliaabssleocrieastuedlts,flsuinocreopthheoyreds.o nInot andedeidtioanny tsotanindtaerndsiotyr cmaeliabsruartieomn.enτtissotfhde orencoirparoncdalaoccfeaplltorartfelus oorfedsecaecntciev,amtioenasoufraenmeexnctisteodf tmheolfelucuolraerscsetantcee, alinfedtiimf aelτifoetfitmhee τd0onisodremteorlmecinueled(fCoFr Pa)rienfetrheennceansyossteecmonwdirthaonugteFaRreEeTx, ptheectdeidffteoregnicveebreetlwiaebelenr1e/sτulatns,dsi1n/cτe0threeflyedctos nthoet rnaeteedoafneynesrtagnydtraarndsoferrcaklEiTbraactcioonrd. iτnigs tthoe reciprocal of all rates of deactivation of an excited molecular state, and if a lifetime τ0 is determined for a reference system without FRET, the difference between

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