Fluorescence Resonance Energy Transfer (FRET)
Practical fluorescence techniques for life scientists”, Helsinki, 2-6.10.2006Graduate course “Fluorescence Resonance Energy Transfer(FRET)Actually seeing molecular proximityYegor Domanov, PhDHelsinki Biophysics and Biomembrane Group, University of Helsinki
Outline What is FRET?– Introduction How does it work?– Physical principles of FRET How is it measured?– Experimental strategies What is it used for?– Applications in biology
Quick facts Powerful tool in biophysics, biochemistry, and cell biology Relies on fluorescence detection, hence:– high sensitivity– modalities: spectroscopy (steady-state, time-resolved, stoppedflow), microscopy, flow cytometry, high-throughput tech.– works well both in vitro and in vivo Requires two: a donor and an acceptor Reports proximity of molecules or moieties within a molecule Works in the range of approx. 1-10 nm
Where it occurs in natureThe antenna complex and photochemical reaction center in a photosystem.Molecular biology of the cell - 4th ed. 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, KeithRoberts, and Peter Walter.
Physical principles of FRET nonradiative weak dipole-dipoleinteraction decays as 1/R 6 no overlap ofelectron clouds spectral shapeunchanged
Kinetics of fluorescence and FRETFörster radius1 R0 kT τD R excited donorlifetime6distance betweenD and A
Förster radius (R0) the distance between D & A at whichthe rate of fluorescence emissionequals the rate of FRET 50% of excited donors will emit light,and 50% will pass energy to acceptors R0 is a function of the donor quant.yield, spectral overlap, and orientation Can be calculated for a given donoracceptor pair from spectroscopic dataR 0 (κ Q D J )216
Förster radiusdepends on the overlap of acceptor absorbancespectrum and donor emission spectrumR 0 (κ Q D J )2 16J FD (λ )ε A (λ )λ4dλ0http://www.olympusfluoview.com 2004-2006 Olympus Corp.
Förster radiusdepends on the mutual orientationof donor and acceptor transition momentsR 0 (κ Q D J )216dynamically averaged value:2κ 32
Typical values of R0DonorAcceptorFörster .3 - 4.1CFTexas Red5.1FluoresceinTetramethylrhodamine4.9 - 5.5Cy3Cy5 5.0GFPYFP5.5 - 5.7BODIPY FLBODIPY FL5.7Rhodamine 6GMalachite Green6.1Cy5Cy5.5 8.0
Efficiency of energy transferR 06kTE 6 1kT τ D R 0 R 6http://www.olympusfluoview.com 2004-2006 Olympus Corp.
Determining the efficiency of FRET by donor intensity:E 1 I DAID by donor fluorescencelifetime:E 1 τ DAτD by acceptor intensity:NBD-PC liposomes à TexasRed-temporin BE ε AC AεD C D I AD 1 IA
FRET microscopy studies: workflow
Applications of FRET Changes: Association, aggregation, conformational changes,enzymatic activity Absolute distances: Structural studies complementary to NMR, EPR,X-ray crystallography, SAS, CryoEM Objects/techniques:– macromolecules (structure, dynamics, biochemical reactions)– supramolecular complexes (multisubunit proteins, polymers,aggregates, prot.-nucl. acid, protein-lipid interactions, membranefusion, lipid rafts)– cellular structures/processes (signalling, transport)– whole cells (expression, viability)
Intramolecular FRET:detecting conformational changeshttp://www.olympusfluoview.com 2004-2006 Olympus Corp.
Example: Internal Movements withinthe 30 S Ribosomal Subunit Single-Cys mutants 13 D-A pairs Alexa 488 à Alexa 568 Association with 50 S subunit Agree with X-ray & cryoEMHickerson et al. (2005) J Mol Biol 354:459 New information unavailablefrom X-ray & cryoEM
Intermolecular FRET:detecting interactions of biomoleculeshttp://www.olympusfluoview.com 2004-2006 Olympus Corp.
Gene-encoded two-component Ca2 indicatorFRET constructs for measuringintracellular calcium. CFP-labeledcalmodulin and YFP-labeledcalmodulin binding peptide (M13YFP) were coexpressed. High Ca2 levels (right) lead to binding andFRET emission of YFP (pseudocolor red); low Ca2 levels (left)lead to little FRET and mostly blueemission (pseudocolor green).The left panel shows two cells before stimulation, while the right panel showsthe same cells after elevation of cytosolic Ca2 by 0.1 mM histamine.Tsien & Miyawaki (1998) Science 280:1954-1955.
FRET in Flow CytomeryFunction-based isolation of novel enzymes from a large libraryProtein variants are displayed on the surfaceof microorganisms and incubated with asynthetic substrate consisting of (1) afluorescent dye (2) a positively chargedmoiety (3) the target scissile bond, and (4) afluorescence resonance energy transfer(FRET) quenching partner. Enzymaticcleavage of the scissile bond results inrelease of the FRET quenching partner whilethe fluorescent product is retained on the cellsurface, allowing isolation of catalyticallyactive clones by fluorescence-activated cellsorting (FACS).NB: Cys/Lys doble labellingOlsen et al. (2000) Nat Biotechnol 18:1071
Genetically encoded FRET reporter ofPKC phosphorylationCKAR is comprised ofmCFP, the FHA2 domain ofRad53p, a PKC substratesequence, and mYFP. Thesubstrate sequence, whenphosphorylated, binds theFHA2 phospho-peptide–binding domain. Thisconformational changeresults in a change in FRET,reversible by phosphatases.Violin et al. (2003) J Cell Biol 161:899-909
Advantages and disadvantagesThe upside. FRET is relatively cheap!!!It is very efficient in measuring changes in distances.You measure distances in molecules in solution.You only need a few µM of labeled proteins.Once you have labeled your molecule, you can have a measurement rapidly.You can measure distances or changes in distances in complex of molecules.and the downside The precision of the measure is impaired by the uncertainty of the orientation factorand by the size of the probes When measuring a change in distance between two probes, the result is a scalar andgive no indications of which probe (donor and/or acceptor) moves. The presence of free labels in solution could mask a change in energy transfer. These measurements give the average distance between the two probes.
Suggested reading P.R. Selvin (2000) The renaissance of fluorescence resonanceenergy transfer. Nat Struct Biol. 7:730-4. P.R. Selvin (1995) Fluorescence resonance energy transfer. MethEnzymol 246:300-334. J.R. Lakowicz (2006) Principles of Fluorescence Spectroscopy, 3rdedn. Springer. Olympus Resource Center: Fluorescence resonance energy transfer(FRET) ions/fretintro.html J. Matko, M. Edidin (1997) Energy transfer methods for detectingmolecular clusters on cell surfaces. Meth Enzymol 278:444-462.
P.R. Selvin (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol.7:730-4. P.R. Selvin (1995) Fluorescence resonance energy transfer. Meth Enzymol246:300-334. J.R. Lakowicz (2006) Principles of Fluorescence Spectroscopy, 3rd edn. Springer. Olympus Resource Center: Fluorescence resonance energy transfer
Introduction What is TR-FRET TR-FRET utilizes time-gated fluorescence intensity measurements to quantitate molecular association or dissociation events. FRET is the non- . Developing a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay 6 Results are in counts/100 flashes. Signal exceeding 1000 counts/flash are saturating the
The FRET image is calculated, and the corresponding histograms are calculated. In the FRET image the FRET efficiency is only calculated for pixels with intensities higher than the threshold. The FRET efficiency color scale is automatically set from 0 to 100% FRET efficiency.
(FRET) Microscopy Mike Lorenz Optical Technology Development mlorenz@mpi-cbg.de FRET-FLIM course, May 2009. 450 500 550 600 650 0.0 0.2 0.4 0.6 0.8 1.0 fluorescence absorption rel. intensity wavelength / nm Quantum yield Ratio of emitted to absorbed photons Stoke'sshift Fluorescence light is always red-
Single-molecule Förster resonance energy trans-fer; Single-pair fluorescence resonance energy transfer Definition Single-molecule fluorescence resonance energy transfer (smFRET)isatechnique usedtomeasure nanometer-scale distances between specific sites on an individual molecule, usually as a function of time. Introduction
fluorescence resonance energy transfer mainly owing to the usage of fluorescence-based probes for FRET applications. In the 1920s, Perrin first introduced the concept of dipole-dipole interaction . Introduction FRET is a collision-free, but distance-dependent photophysical process where radiationless transfer of energy occurs from an .
INTRODUCTION Fluorescence resonance energy transfer (FRET) microscopy can be used to visualize the spatial and temporal localization of interactions between labeled proteins within living cells (Kraynov et al., 2000). FRET occurs when an excited donor fluorophore is within the critical transfer distance (;30-100
1. Introduction: fluorescent proteins (FPs) and fluorescence resonance energy transfer (FRET) 2. The engineering of FRET biosensors 3. The application of FRET biosensors i. Subcellular and microdomain imaging ii. Multi-color and Correlation imaging of molecular hierarchies iii. Visualize how cells perceive environmental physical cues
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