FRET – Molecular FRET – Molecular StorytellingStorytelling

April 22, 2005April 22, 2005

Rose ChaseRose Chase

The beginning…The beginning…

Theodor FTheodor Försterörster 1940s proposed mathematical law for dependence of 1940s proposed mathematical law for dependence of

fluorescence decay of donor (D) on the concentration fluorescence decay of donor (D) on the concentration of acceptor (A), assuming a dipole-dipole interaction of acceptor (A), assuming a dipole-dipole interaction in solution (J.Phys.Chem. 1965, 69, 1061-1062)in solution (J.Phys.Chem. 1965, 69, 1061-1062)

Exponential decay for one D molecule

But…But…

One D, not applicable, therefore average One D, not applicable, therefore average over all distances over all distances

His equations, although eventually proven His equations, although eventually proven correct, were founded on correct, were founded on incorrect/incomplete assumptionsincorrect/incomplete assumptionsDifferent distributions of ADifferent distributions of AMathematical shortcutsMathematical shortcutsBut THREE different scientists came to same But THREE different scientists came to same

conclusion nonethelessconclusion nonetheless

Missing pointMissing point

ASSUMINGASSUMINGOnce an A is excited, no further quenching of Once an A is excited, no further quenching of

DDTherefore, A molecules decay exponentiallyTherefore, A molecules decay exponentially ““It thus seems, that the proper result obtained It thus seems, that the proper result obtained

by Fby Förster nevertheless does not justify his örster nevertheless does not justify his statistical reasoning.” -Mira Leibowitzstatistical reasoning.” -Mira Leibowitz

Impact of FRETImpact of FRET

After the initial fuss about the validity of FAfter the initial fuss about the validity of Förster’s örster’s derivations, little else.derivations, little else.

Resurgence in past several years, especially in Resurgence in past several years, especially in biology/biochemistry/biophysics thanks to:biology/biochemistry/biophysics thanks to: FRET capable spectral GFP mutantsFRET capable spectral GFP mutants Engineering of peptides w/novel fluorescent reagentsEngineering of peptides w/novel fluorescent reagents Ability to couple to different imaging techniquesAbility to couple to different imaging techniques AND the need to “see” finer detailsAND the need to “see” finer details

Power of FRETPower of FRET

Probe macromolecular interactionsProbe macromolecular interactions Interaction assumed upon fluorescence decayInteraction assumed upon fluorescence decay

Study kinetics of association/dissociation Study kinetics of association/dissociation between macromoleculesbetween macromolecules

Estimation of distancesEstimation of distances In vitro AND in vivoIn vitro AND in vivoSingle molecule studiesSingle molecule studies

What is FRET?What is FRET?

FFörster Resonance Energy Transferörster Resonance Energy TransferFluorescence if both D and A are fluorescentFluorescence if both D and A are fluorescent

Radiation-less energy transition b/t a D Radiation-less energy transition b/t a D and A w/ finite probability based on and A w/ finite probability based on proximityproximity

Energy is transferred through the resonant Energy is transferred through the resonant coupling of the dipole moments of D and Acoupling of the dipole moments of D and A

FFöörster Resonance Energy rster Resonance Energy TransferTransfer

-Complicated, but thorough energy diagram, depicting all possible paths of relaxationupon excitation

FRETFRET

More simple, concise More simple, concise scheme, depicting the scheme, depicting the transfer of energy transfer of energy from D to Afrom D to A

Effects of FRETEffects of FRET

Intensity of D decreasesIntensity of D decreasesSensitized fluorescence of A appears Sensitized fluorescence of A appears

upon D excitationupon D excitationLifetime of D excited stated decreasesLifetime of D excited stated decreasesPolarization anisotropy increasesPolarization anisotropy increasesTaking advantage of these points…Taking advantage of these points…

Curr. Opin. Immun. 2004, 16, 418-427Curr. Opin. Immun. 2004, 16, 418-427

Efficiency (E)Efficiency (E)

Measuring FRET Measuring FRET efficiencyefficiency

These methods are These methods are best for fixed cellsbest for fixed cells

Photobleaching Photobleaching usually done with high usually done with high power laser setting in power laser setting in confocalconfocal

Spectral MethodsSpectral Methods

Goal of spectral methods is to selectively measure FRET Goal of spectral methods is to selectively measure FRET from D, A, or sensitized fluorescencefrom D, A, or sensitized fluorescence

Problem – Artifacts from sequential acquisition of images Problem – Artifacts from sequential acquisition of images unless simultaneousunless simultaneous

Fluorescence Lifetime Imaging Fluorescence Lifetime Imaging (FLIM)(FLIM)

Great for biological Great for biological samples b/c decay of D samples b/c decay of D increases w/FRETincreases w/FRET

Time/Frequency domain Time/Frequency domain measurements measurements

Time - can get both Time - can get both degree of complex degree of complex formation and intrinsic Eformation and intrinsic E

FLIM + 2 photon FLIM + 2 photon excitation + FRET excitation + FRET in in vivo monitoringvivo monitoring

Polarization Anisotropy and FRETPolarization Anisotropy and FRET

Polarization anisotropy – ability of fluorescence Polarization anisotropy – ability of fluorescence to retain the direction of polarization of excitation to retain the direction of polarization of excitation sourcesource

Illuminate sample w/ polarized excitation and Illuminate sample w/ polarized excitation and read fluorescence parallel and perpendicular to read fluorescence parallel and perpendicular to polarization axispolarization axis

Ex. GFP – slow moving w/ short lifetime, Ex. GFP – slow moving w/ short lifetime, therefore little loss therefore little loss parallel; but cluster and parallel; but cluster and get energy migration FRET get energy migration FRET perpendicular perpendicular

Limits Limits

ASSUMPTION of interaction based on ASSUMPTION of interaction based on fluorescence, possible intermediatefluorescence, possible intermediate

Changes in E may be attributed to Changes in E may be attributed to conformational changes or restrictionsconformational changes or restrictions

Crowding/aggregationCrowding/aggregationCOSTCOSTOn to applications…On to applications…

Ratiometric indicator for ClRatiometric indicator for Cl--

Neuron 2000, 27, 447-459Neuron 2000, 27, 447-459ClCl--

Regulation of cell volume, intracell. pH, fluid Regulation of cell volume, intracell. pH, fluid secretion, stabilization of resting potentialsecretion, stabilization of resting potential

Developmental intracell. [ClDevelopmental intracell. [Cl--] determines ] determines whether GAGAergic synapes excite/inhibit whether GAGAergic synapes excite/inhibit postsynaptic targetpostsynaptic target

Change in gradient has other periphery Change in gradient has other periphery effectseffects

Effects of incorrect [Cl-]Effects of incorrect [Cl-] Cystic fibrosis – thick and sticky sweat, mucus, Cystic fibrosis – thick and sticky sweat, mucus,

saliva, and digestive juicessaliva, and digestive juices Myotonia congenita – slow relaxation of musclesMyotonia congenita – slow relaxation of muscles Inherited hypercalciuric nephrolithiasis – kidney Inherited hypercalciuric nephrolithiasis – kidney

stones (exacerbate w/high salt)stones (exacerbate w/high salt) Bartter syndrome – muscle cramping and Bartter syndrome – muscle cramping and

weakness, constipation, increases freq. of weakness, constipation, increases freq. of urination, and growth failure (characteristic low urination, and growth failure (characteristic low blood [Clblood [Cl--], high urine [Cl], high urine [Cl--])])

Hyperekplexia/startle disease – excessive startle Hyperekplexia/startle disease – excessive startle reaction, extreme muscle tension, unstable gaitreaction, extreme muscle tension, unstable gait

Measuring [Cl-]Measuring [Cl-]

While using FRET realized ClWhile using FRET realized Cl-- senisitivity! senisitivity!Fusion protein CPF/YFP fluorophores can Fusion protein CPF/YFP fluorophores can

be used as a ratiometric, targetable, and be used as a ratiometric, targetable, and genetically encoded indicatorgenetically encoded indicator

On to the good stuff… On to the good stuff…

Checking the proteinChecking the protein All excited at 434nm All excited at 434nm

and normalized to and normalized to peaks at 527peaks at 527

Depict Ratio of Depict Ratio of YFP/CFP emissionYFP/CFP emission

Check for FRET by Check for FRET by TEV cleavage TEV cleavage increase of D at increase of D at 485nm, loss of A at 485nm, loss of A at 527nm 527nm

Ion sensitivity Ion sensitivity

A - Halide preferenceA - Halide preference B - inc. quenching at B - inc. quenching at

inc. [Hinc. [H++]] C - re-plot as fxn of C - re-plot as fxn of

pHpH D - 10 fold shift for D - 10 fold shift for

1pH unit1pH unit E - effect on apparent E - effect on apparent

[Cl[Cl--]] D - %errorD - %error

Calibrating ClomeleonCalibrating Clomeleon

RRmaxmax and R and Rminmin using using

patch pipettepatch pipette [Cl[Cl--] = Kd * ] = Kd * (2.39 – R)(2.39 – R)

(R – 0.49)(R – 0.49)

Utilizing ClomeleonUtilizing Clomeleon

In embryonic In embryonic neurons, GABA elicits neurons, GABA elicits depol., in adults, depol., in adults, hyperpol.hyperpol.

Switch is paralleled Switch is paralleled by changes in [Clby changes in [Cl--]]

Use clomeleon to Use clomeleon to directly measure!directly measure! Resting [ClResting [Cl--] in living ] in living

cellscells

GABA mediationGABA mediation A & B – pulse of GABA A & B – pulse of GABA

via micropipette, 10-30via micropipette, 10-30μμm m from soma of individual from soma of individual neuronneuron Simultaneous whole cell Simultaneous whole cell

patch clamp and patch clamp and fluorescencefluorescence

C – good fit of conc. to C – good fit of conc. to currentcurrent

D – discrepancy from D – discrepancy from removal of Clremoval of Cl-- from cell from cell

E – pH dependence E – pH dependence checkcheck

Dendritic SignalingDendritic Signaling

Concentration and current monitoredConcentration and current monitored Change in conc. certainly enough to induce switch of Change in conc. certainly enough to induce switch of

GABAergic synaptic input from inhibitory to excitatoryGABAergic synaptic input from inhibitory to excitatory

Perks of ClomeleonPerks of Clomeleon

Visible light excitationVisible light excitation Good signal to noise, even at low conc.Good signal to noise, even at low conc. Not affected by other physiologically relevant Not affected by other physiologically relevant

anionsanions Simple loadingSimple loading High MW retards diffusion, preventing collapse High MW retards diffusion, preventing collapse

of spatial gradients also retards wash out during of spatial gradients also retards wash out during recordingrecording

Genetically encoded Genetically encoded specificity/targeting specificity/targeting

Probing interactions with FRETProbing interactions with FRET

Biochemistry 2004, 43, 8754-8765Biochemistry 2004, 43, 8754-8765Phospholamban (PLB) inhibits Ca-ATPase Phospholamban (PLB) inhibits Ca-ATPase

of the sarcoplasmic reticulum (SERCA) at of the sarcoplasmic reticulum (SERCA) at submicromolar Casubmicromolar Ca+2+2

Inhibition relieved by either elevation of Inhibition relieved by either elevation of CaCa+2+2 to micromolar, or phosphorylation of to micromolar, or phosphorylation of PLB (adrenergic response)PLB (adrenergic response)

Importance of PLB-SERCAImportance of PLB-SERCA

Linked to development of dilated Linked to development of dilated cardiomyopathy and progression to heart cardiomyopathy and progression to heart failure in young adultsfailure in young adults

PLB possible drug targetPLB possible drug targetTherefore, elucidation of mechanism by Therefore, elucidation of mechanism by

which PLB regulates SERCA importantwhich PLB regulates SERCA important

The goalThe goal

Decreased inhibition Decreased inhibition maybe due to PLB maybe due to PLB dissoc. from SERCA, dissoc. from SERCA, must measure Kmust measure Kd2d2

Donor labeled Donor labeled SERCA and Acceptor SERCA and Acceptor labeled PLB in labeled PLB in reconstituted reconstituted membranesmembranes

A quick look at methodsA quick look at methods

PLBPLBDABCYL-SE, chosen b/c lacks fluorescence DABCYL-SE, chosen b/c lacks fluorescence

emission and therefore does not interfere with emission and therefore does not interfere with donor emission signaldonor emission signal

SERCASERCALabeled with IAEDANSLabeled with IAEDANS

Excitation at 351.1nmExcitation at 351.1nmTemp controlled by water bath at 25Temp controlled by water bath at 25°C°C

A tiny bit of math…A tiny bit of math… A lot like earlier equations…A lot like earlier equations… FRET Efficiency and D-A distanceFRET Efficiency and D-A distance

High and Low Ca+2 FxnHigh and Low Ca+2 Fxn

Reconstituted, Reconstituted, labeled SERCA is labeled SERCA is functional and fully functional and fully inhibited at high Cainhibited at high Ca+2+2, , and nearly fully and nearly fully functional at low Cafunctional at low Ca+2+2

Labeled PLB also Labeled PLB also functions same as functions same as unlabeledunlabeled

Supporting interaction b/t SERCA & Supporting interaction b/t SERCA & PLBPLB

At distance greater than 45At distance greater than 45Å, FRET less than 10%Å, FRET less than 10% AND b/c SERCA ~55-90Å, FRET only detectable if AND b/c SERCA ~55-90Å, FRET only detectable if

proteins physically interact in membrane, with rapid proteins physically interact in membrane, with rapid FRET dissipation w/ PLB dissociationFRET dissipation w/ PLB dissociation

At low pCaAt low pCa+2+2

At high pCaAt high pCa+2+2, much the same, much the same

Small but significant (and ONLY) difference was in maximal Small but significant (and ONLY) difference was in maximal energy transfer energy transfer

D-A distance at low 33.1+0.4D-A distance at low 33.1+0.4Å vs. 34.2+0.2Å at high, Å vs. 34.2+0.2Å at high, therefore small structural change, but no dissociationtherefore small structural change, but no dissociation

Time resolved FRETTime resolved FRET

Supports steady state data at high and low Supports steady state data at high and low CaCa+2+2

More ATPase activityMore ATPase activity

Independent of Independent of SERCA conc., SERCA conc., dependent on total dependent on total PLB/SERCA ratio, PLB/SERCA ratio, membrane and freemembrane and free

Inhibition increase Inhibition increase with addition of PLB with addition of PLB indicates nonspecific indicates nonspecific component of component of inhibitioninhibition

Anisotropy MeasurementsAnisotropy Measurements

PLB increases anisotropy, indicating SERCA aggregationPLB increases anisotropy, indicating SERCA aggregation May account for fig.10May account for fig.10

Therefore…Therefore…

The conclude the hypothesis must be The conclude the hypothesis must be revised.revised.

Conclusions Conclusions

Affinity of PLB for SERCA is so high it is Affinity of PLB for SERCA is so high it is essentially a SERCA subunit under essentially a SERCA subunit under physiological conditionsphysiological conditions

Relief of inhibition at micromolar CaRelief of inhibition at micromolar Ca+2+2 is is due to structural rearrangement within the due to structural rearrangement within the SERCA-PLB complex, rather than SERCA-PLB complex, rather than dissociationdissociation

Association of TM helices in lipid Association of TM helices in lipid bilayersbilayers

Langmuir 2004, 20, 9053-9060Langmuir 2004, 20, 9053-9060 Membrane proteinsMembrane proteins

Cell adhesionCell adhesion RecognitionRecognition MotilityMotility Energy productionEnergy production Transport of nutrients and cholesterolTransport of nutrients and cholesterol Biochemical signal transductionBiochemical signal transduction

Engineering of surface immobilized bilayers Engineering of surface immobilized bilayers containing active, integral membrane proteins:containing active, integral membrane proteins: Drug screening devices, mimetics of cell and tissue Drug screening devices, mimetics of cell and tissue

surfaces, matrices for stress free cell immobilizationsurfaces, matrices for stress free cell immobilization

Focus on…Focus on…

Fibroblast growth factor receptor 3 Fibroblast growth factor receptor 3 (FGFR3)(FGFR3)Causes various cancers and developmental Causes various cancers and developmental

abnormalities by affecting lateral dimerization abnormalities by affecting lateral dimerization in the membranein the membrane

AmbitionsAmbitions

Characterize the architecture of surface Characterize the architecture of surface supported protein/lipid bilayerssupported protein/lipid bilayers Fluorescence recovery after photobleaching (FRAP)Fluorescence recovery after photobleaching (FRAP) FRET – show that FRET signal is same in suspended FRET – show that FRET signal is same in suspended

liposomes and in surface supported bilayersliposomes and in surface supported bilayers

Further develop an imaging FRET methodology Further develop an imaging FRET methodology that may provide a less expensive and less that may provide a less expensive and less tedious alternative to solution FRETtedious alternative to solution FRET

Excitation and structure checkExcitation and structure check

A – FRET% A – FRET% calculated from:calculated from:

B – CD spectra to B – CD spectra to check for helix check for helix structurestructure

Short on PhotobleachingShort on Photobleaching

Images taken of D and A prior to bleachingImages taken of D and A prior to bleachingA is bleached in small area, while A is bleached in small area, while

measuring the decrease in intensity. measuring the decrease in intensity. Continued until no significant change in Continued until no significant change in intensity is noted.intensity is noted.

D image captured after A bleaching. With D image captured after A bleaching. With A bleached, no ET from D to A. FRET is A bleached, no ET from D to A. FRET is obvious from the appearance of a bright obvious from the appearance of a bright spot in the D spot, where A was bleached. spot in the D spot, where A was bleached.

More photobleaching…More photobleaching…

Did run control to ensure A bleaching did not bleach DDid run control to ensure A bleaching did not bleach D

FRAPFRAP

Small area is bleached, fluorescence Small area is bleached, fluorescence monitored. If molecules are mobile, monitored. If molecules are mobile, bleached spot will gradually disappear due bleached spot will gradually disappear due to lateral diffusion of both bleached and to lateral diffusion of both bleached and fluorescent moleculesfluorescent molecules

More FRAPMore FRAP

Less pretty view…Less pretty view…

FRAP indicates…FRAP indicates…

Substrate immobilizes the proteins, but Substrate immobilizes the proteins, but does not induce protein dissolution from does not induce protein dissolution from the lipid matrixthe lipid matrix

Protein-protein interactions in supported Protein-protein interactions in supported bilayer are like frozen “snapshot” of bilayer are like frozen “snapshot” of interactions in free suspended vesiclesinteractions in free suspended vesicles

Solution FRET vs. Imaging FRETSolution FRET vs. Imaging FRET

More FRET resultsMore FRET results

ConclusionsConclusions

Imaging FRET can be used as a means Imaging FRET can be used as a means to quantify TM helix dimerization in surface to quantify TM helix dimerization in surface supported bilayerssupported bilayers

Better, due to use of a single sample Better, due to use of a single sample repeatedlyrepeatedly

Less expensive than solution (equipment Less expensive than solution (equipment and sample volume)and sample volume)