FRET – Molecular
Storytelling
April 22, 2005
Rose Chase
The beginning…

Theodor Förster

1940s proposed mathematical law for dependence of
fluorescence decay of donor (D) on the concentration
of acceptor (A), assuming a dipole-dipole interaction
in solution (J.Phys.Chem. 1965, 69, 1061-1062)
Exponential decay for one D molecule
But…
One D, not applicable, therefore average
over all distances
 His equations, although eventually proven
correct, were founded on
incorrect/incomplete assumptions

Different distributions of A
 Mathematical shortcuts
 But THREE different scientists came to same
conclusion nonetheless

Missing point

ASSUMING
Once an A is excited, no further quenching of
D
 Therefore, A molecules decay exponentially
 “It thus seems, that the proper result obtained
by Förster nevertheless does not justify his
statistical reasoning.” -Mira Leibowitz

Impact of FRET


After the initial fuss about the validity of Förster’s
derivations, little else.
Resurgence in past several years, especially in
biology/biochemistry/biophysics thanks to:



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FRET capable spectral GFP mutants
Engineering of peptides w/novel fluorescent reagents
Ability to couple to different imaging techniques
AND the need to “see” finer details
Power of FRET

Probe macromolecular interactions

Interaction assumed upon fluorescence decay
Study kinetics of association/dissociation
between macromolecules
 Estimation of distances
 In vitro AND in vivo
 Single molecule studies

What is FRET?

Förster Resonance Energy Transfer

Fluorescence if both D and A are fluorescent
Radiation-less energy transition b/t a D
and A w/ finite probability based on
proximity
 Energy is transferred through the resonant
coupling of the dipole moments of D and A

Förster Resonance Energy
Transfer
-Complicated, but thorough
energy diagram, depicting
all possible paths of relaxation
upon excitation
FRET

More simple, concise
scheme, depicting the
transfer of energy
from D to A
Effects of FRET
Intensity of D decreases
 Sensitized fluorescence of A appears upon
D excitation
 Lifetime of D excited stated decreases
 Polarization anisotropy increases
 Taking advantage of these points…


Curr. Opin. Immun. 2004, 16, 418-427
Efficiency (E)



Measuring FRET
efficiency
These methods are
best for fixed cells
Photobleaching
usually done with high
power laser setting in
confocal
Spectral Methods


Goal of spectral methods is to selectively measure FRET
from D, A, or sensitized fluorescence
Problem – Artifacts from sequential acquisition of images
unless simultaneous
Fluorescence Lifetime Imaging
(FLIM)




Great for biological
samples b/c decay of D
increases w/FRET
Time/Frequency domain
measurements
Time - can get both
degree of complex
formation and intrinsic E
FLIM + 2 photon
excitation + FRET  in
vivo monitoring
Polarization Anisotropy and FRET



Polarization anisotropy – ability of fluorescence
to retain the direction of polarization of excitation
source
Illuminate sample w/ polarized excitation and
read fluorescence parallel and perpendicular to
polarization axis
Ex. GFP – slow moving w/ short lifetime,
therefore little loss  parallel; but cluster and
get energy migration FRET  perpendicular
Limits
ASSUMPTION of interaction based on
fluorescence, possible intermediate
 Changes in E may be attributed to
conformational changes or restrictions
 Crowding/aggregation
 COST
 On to applications…

Ratiometric indicator for ClNeuron 2000, 27, 447-459
 Cl
Regulation of cell volume, intracell. pH, fluid
secretion, stabilization of resting potential
 Developmental intracell. [Cl-] determines
whether GAGAergic synapes excite/inhibit
postsynaptic target
 Change in gradient has other periphery
effects

Effects of incorrect [Cl-]


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Cystic fibrosis – thick and sticky sweat, mucus,
saliva, and digestive juices
Myotonia congenita – slow relaxation of muscles
Inherited hypercalciuric nephrolithiasis – kidney
stones (exacerbate w/high salt)
Bartter syndrome – muscle cramping and
weakness, constipation, increases freq. of
urination, and growth failure (characteristic low
blood [Cl-], high urine [Cl-])
Hyperekplexia/startle disease – excessive startle
reaction, extreme muscle tension, unstable gait
Measuring [Cl-]
While using FRET realized Cl- senisitivity!
 Fusion protein CPF/YFP fluorophores can
be used as a ratiometric, targetable, and
genetically encoded indicator
 On to the good stuff…

Checking the protein



All excited at 434nm
and normalized to
peaks at 527
Depict Ratio of
YFP/CFP emission
Check for FRET by
TEV cleavage 
increase of D at
485nm, loss of A at
527nm
Ion sensitivity

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A - Halide preference
B - inc. quenching at
inc. [H+]
C - re-plot as fxn of
pH
D - 10 fold shift for
1pH unit
E - effect on apparent
[Cl-]
D - %error
Calibrating Clomeleon


Rmax and Rmin using
patch pipette
[Cl-] = Kd * (2.39 – R)
(R – 0.49)
Utilizing Clomeleon



In embryonic
neurons, GABA elicits
depol., in adults,
hyperpol.
Switch is paralleled
by changes in [Cl-]
Use clomeleon to
directly measure!

Resting [Cl-] in living
cells
GABA mediation

A & B – pulse of GABA
via micropipette, 1030μm from soma of
individual neuron



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Simultaneous whole cell
patch clamp and
fluorescence
C – good fit of conc. to
current
D – discrepancy from
removal of Cl- from cell
E – pH dependence
check
Dendritic Signaling


Concentration and current monitored
Change in conc. certainly enough to induce switch of
GABAergic synaptic input from inhibitory to excitatory
Perks of Clomeleon


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Visible light excitation
Good signal to noise, even at low conc.
Not affected by other physiologically relevant
anions
Simple loading
High MW retards diffusion, preventing collapse
of spatial gradients also retards wash out during
recording
Genetically encoded  specificity/targeting
Probing interactions with FRET
Biochemistry 2004, 43, 8754-8765
 Phospholamban (PLB) inhibits Ca-ATPase
of the sarcoplasmic reticulum (SERCA) at
submicromolar Ca+2
 Inhibition relieved by either elevation of
Ca+2 to micromolar, or phosphorylation of
PLB (adrenergic response)

Importance of PLB-SERCA
Linked to development of dilated
cardiomyopathy and progression to heart
failure in young adults
 PLB possible drug target
 Therefore, elucidation of mechanism by
which PLB regulates SERCA important

The goal


Decreased inhibition
maybe due to PLB
dissoc. from SERCA,
must measure Kd2
Donor labeled
SERCA and Acceptor
labeled PLB in
reconstituted
membranes
A quick look at methods

PLB


DABCYL-SE, chosen b/c lacks fluorescence
emission and therefore does not interfere with
donor emission signal
SERCA

Labeled with IAEDANS
Excitation at 351.1nm
 Temp controlled by water bath at 25°C

A tiny bit of math…


A lot like earlier equations…
FRET Efficiency and D-A distance
High and Low Ca+2 Fxn


Reconstituted,
labeled SERCA is
functional and fully
inhibited at high Ca+2,
and nearly fully
functional at low Ca+2
Labeled PLB also
functions same as
unlabeled
Supporting interaction b/t SERCA &
PLB


At distance greater than 45Å, FRET less than 10%
AND b/c SERCA ~55-90Å, FRET only detectable if
proteins physically interact in membrane, with rapid
FRET dissipation w/ PLB dissociation
At low pCa+2
At high pCa+2, much the same


Small but significant (and ONLY) difference was in maximal
energy transfer
D-A distance at low 33.1+0.4Å vs. 34.2+0.2Å at high,
therefore small structural change, but no dissociation
Time resolved FRET

Supports steady state data at high and low
Ca+2
More ATPase activity


Independent of
SERCA conc.,
dependent on total
PLB/SERCA ratio,
membrane and free
Inhibition increase
with addition of PLB
indicates nonspecific
component of
inhibition
Anisotropy Measurements

PLB increases anisotropy, indicating SERCA aggregation

May account for fig.10
Therefore…

The conclude the hypothesis must be
revised.
Conclusions
Affinity of PLB for SERCA is so high it is
essentially a SERCA subunit under
physiological conditions
 Relief of inhibition at micromolar Ca+2 is
due to structural rearrangement within the
SERCA-PLB complex, rather than
dissociation

Association of TM helices in lipid
bilayers


Langmuir 2004, 20, 9053-9060
Membrane proteins

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Cell adhesion
Recognition
Motility
Energy production
Transport of nutrients and cholesterol
Biochemical signal transduction
Engineering of surface immobilized bilayers
containing active, integral membrane proteins:

Drug screening devices, mimetics of cell and tissue
surfaces, matrices for stress free cell immobilization
Focus on…

Fibroblast growth factor receptor 3
(FGFR3)

Causes various cancers and developmental
abnormalities by affecting lateral dimerization
in the membrane
Ambitions

Characterize the architecture of surface
supported protein/lipid bilayers



Fluorescence recovery after photobleaching (FRAP)
FRET – show that FRET signal is same in suspended
liposomes and in surface supported bilayers
Further develop an imaging FRET methodology
that may provide a less expensive and less
tedious alternative to solution FRET
Excitation and structure check

A – FRET%
calculated from:

B – CD spectra to
check for helix
structure
Short on Photobleaching
Images taken of D and A prior to bleaching
 A is bleached in small area, while
measuring the decrease in intensity.
Continued until no significant change in
intensity is noted.
 D image captured after A bleaching. With
A bleached, no ET from D to A. FRET is
obvious from the appearance of a bright
spot in the D spot, where A was bleached.

More photobleaching…

Did run control to ensure A bleaching did not bleach D
FRAP

Small area is bleached, fluorescence
monitored. If molecules are mobile,
bleached spot will gradually disappear due
to lateral diffusion of both bleached and
fluorescent molecules
More FRAP
Less pretty view…
FRAP indicates…
Substrate immobilizes the proteins, but
does not induce protein dissolution from
the lipid matrix
 Protein-protein interactions in supported
bilayer are like frozen “snapshot” of
interactions in free suspended vesicles

Solution FRET vs. Imaging FRET
More FRET results
Conclusions
Imaging FRET can be used as a means
to quantify TM helix dimerization in surface
supported bilayers
 Better, due to use of a single sample
repeatedly
 Less expensive than solution (equipment
and sample volume)
