Johan Hofkens Laboratory of Photochemistry and Spectroscopy Katholieke Universiteit Leuven - Belgium...

Johan Hofkens

Laboratory of Photochemistry and SpectroscopyKatholieke Universiteit Leuven - Belgium

K.U.LEUVEN

Theories and methods to study molecular interactions :

fluorescence and it’s applications

30/11/2005 : Basic principles of fluorescence- absorption, emission, charateristics of a probe- time resolved measurements- quenching, anisotropy- energy transfer, electron transfer- examples

02/12/2005 : Fluorescence microscopy (Dr J. Hotta)- definitions, parameters- different types of microscopy

07/12/2005 : Single molecule fluorescence microscopy- why single molecule studies- different single molecule approaches

09/12/2005 : Applications of fluorescence microscopy

Principles of fluorescence and it’s applications to studymolecular interactions

Fluorescence

• What is it?

• Where does it come from?

• Parameters, Advantages, Techniques

• Examples

http://www.chem.kuleuven.ac.be/research/mds/bioinformatics_courses.htm

References & additional reading

For light to be useful to us it must interact with matter

• Types of interaction:– Reflection– Refraction – Absorption (followed by emission)

Fluorescence : photons emitted by organic molecules after interaction with light

Dual Nature of light: wave and particle

– Light as a wave:

= c/ E = h = hc/

Dual Nature of light: wave and particle

– Light as a particle:

Visible light– Why do we call this “visible” light

Wavelength Range

(nanometers)Perceived Color

340-400Near Ultraviolet (UV;

Invisible)400-430 Violet

430-500 Blue

500-560 Green

560-620 Yellow to Orange

620-700 Orange to Red

Over 700 Near Infrared (IR; Invisible)

Overview of electromagnetic radiation

Absorption : electronic transition(s) in a molecule

Orbitals, molecular orbitals

Simplified Jablonski Diagram

n er g

hvex hvem

Return to ground state results in emission of radiation (fluorochrome).

Absorption of photon elevates chromophore to excited state.

Absorption : Franck Condon Principal, Vibrational fine structure

Characteristics of stationary molecular fluorescence

- Effect on emission is similar as for absorption- For rigid molecules with little displacement between PES mirror symmetry and large overlap

- 0 . 4- 0 . 3

- 0 . 2- 0 . 1

0 . 00 . 1

0 . 20 . 3

0 . 40 . 0

Q - Q e ( i n Å )

E ( i n e V )

24000 22320 20640 18960 172800E+0

1E+6 00

030201

Intensity a.u.

Wavenumber cm

- Effect on emission is similar as for absorption- For rigid molecules with displacement between PES mirror symmetry and small overlap

-0.4-0.3

-0.2-0.1

0.00.1

0.20.3

0.40.0

r ( i n Å )

E ( i n e V )

24000 22320 20640 18960 172800E+0

1,5E+4

2,5E+4

3,5E+4

Intensity a.u.

Wavenumber cm-1

- Effect on emission is similar as for absorption- For rigid molecules with displacement between PES mirror symmetry and small overlap

9-Cyanoanthracene in methanol

300 350 400 450 500 550 6000

Wavelength (nm)

Absorbance Relative Intensity (a.u.)

0-10-2

G° = 24480 cm-1

s = 640 cm-1

h = 1560 cm-1

= 1290 cm-1

- Repulsive S1 PES results in a broad unstructured spectrum.- Maximum given by the AB line.- Symmetric (Gaussian) absorption band.

- Repulsive ground state, emission will result in a broad band- When stabilizing excited state interaction is caused by two identical molecules it is called excimer, when the interaction is caused by two different molecules it is called exciplex.

h-Mh-EEele

2,5 3 3,5 4 4,50

Distance (Å)

Energy (eV)

Stokes shift– is the energy difference between the lowest

energy peak of absorbence and the highest energy of emission

495 nm 520 nm

Stokes Shift is 25 nmFluoresceinmolecule

Wavelength

result of : vibrational relaxation solvent reorganization

Stokes shift

Fluorophores/chromophores/probes

• Chromophores are compounds or molecules which absorb light

• They contain generally aromatic rings

• The longer the conjugated system, the longer wavelength of fluorescence.

Fluorophores/chromophores/probes

Allophycocyanin (APC)Protein 632.5 nm (HeNe)

Excitation Emisson

300 nm 400 nm 500 nm 600 nm 700 nm

Excitation - Emission Peaks

Fluorophore EXpeak EM peak

% Max Excitation at488 568 647 nm

FITC 496 518 87 0 0Bodipy 503 511 58 1 1Tetra-M-Rho 554 576 10 61 0L-Rhodamine 572 590 5 92 0Texas Red 592 610 3 45 1CY5 649 666 1 11 98

Probes for Proteins

FITC 488 525

PE 488 575

APC 630 650

PerCP™ 488 680

Cascade Blue 360 450

Coumerin-phalloidin 350 450

Texas Red™ 610 630

Tetramethylrhodamine-amines 550 575

CY3 (indotrimethinecyanines) 540 575

CY5 (indopentamethinecyanines) 640 670

Probe Excitation Emission

• Hoechst 33342 (AT rich) (uv)346 460• DAPI (uv) 359 461• POPO-1 434 456• YOYO-1 491 509• Acridine Orange (RNA) 460 650• Acridine Orange (DNA) 502 536• Thiazole Orange (vis) 509 525• TOTO-1 514 533• Ethidium Bromide 526 604• PI (uv/vis) 536 620• 7-Aminoactinomycin D (7AAD) 555 655

Probes for Nucleic Acids

DNA Probes• AO

– Metachromatic dye• concentration dependent emission• double stranded NA - Green• single stranded NA - Red

• AT/GC binding dyes– AT rich: DAPI, Hoechst, quinacrine

– GC rich: antibiotics bleomycin, chromamycin A3, mithramycin, olivomycin, rhodamine 800

Probes for Ions

• INDO-1 Ex350Em405/480

• QUIN-2 Ex350 Em490

• Fluo-3 Ex488 Em525

• Fura -2 Ex330/360 Em510

pH Sensitive Indicators

• SNARF-1 488 575

• BCECF 488 525/620

440/488 525[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]

Probe Excitation Emission

Probes for Oxidation States

• DCFH-DA(H2O2) 488 525

• HE (O2-) 488 590

• DHR 123 (H2O2) 488 525

Probe Oxidant Excitation Emission

DCFH-DA - dichlorofluorescin diacetateHE - hydroethidineDHR-123 - dihydrorhodamine 123

Specific Organelle Probes

BODIPY Golgi 505 511

NBD Golgi 488 525

DPH Lipid 350 420

TMA-DPH Lipid 350 420

Rhodamine 123 Mitochondria 488 525

DiO Lipid 488 500

diI-Cn-(5) Lipid 550 565

diO-Cn-(3) Lipid 488 500

Probe Site Excitation Emission

BODIPY - borate-dipyrromethene complexesNBD - nitrobenzoxadiazoleDPH - diphenylhexatrieneTMA - trimethylammonium

Other Probes of Interest

• GFP - Green Fluorescent Protein– GFP is from the chemiluminescent jellyfish Aequorea

victoria

– excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm

– contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence

– Major application is as a reporter gene for assay of promoter activity

– requires no added substrates

Excited State Dynamics of the Green Fluorescent Proteins

Wild-type GFP

Serine65

Arginine96

Glutamine94

Tyrosine66

Glu222

Glycine67

Phenylalanine64

Histidine148

Excited State Dynamics of the Green Fluorescent Proteins

•monitoring proteins, organelles, cells in living tissue.

•protein-protein interaction using double labeling and FRET.

•membrane traffic studies.

•pH sensor.

•Ca2+ sensor.

•……….

Applications :

Fluorescent proteins

DsRed – a longer wavelength substitute for GFPs

New trends in GFP-research

• Optical marking (following intracellular dynamics) or kindling

Patterson, G. H. & Lippincott-Schwartz, J. Science 2002, 297, 1873.

Photo-Switchable Fluorescent Protein Dronpa

• Dronpa is a monomeric GFP-like fluorescent protein from coral Echinophyllia sp.

• Dronpa shows reversible photoswitching on irradiation with a 488 nm and 405 nm light.

488 nm

405 nm

Steady-State Spectra of Dronpa

pH = 7.4 pH = 5.0

• Deprotonated form (B form); fluorescent state, fl488 = 0.85, fl = 3.6 ns

• Protonated form (A1 form); dim state, fl390 = 0.02, fl = 14 ps

300 400 500 600

Wavelength / nm300 400 500 600

Wavelength / nm

300 400 500 6000.00

Wavelength / nm

Photoswitching of Dronpa at the Ensemble Level

488 nm

405 nm

300 400 500 6000.00

Wavelength / nm

0 300 600 900 12000.00

ceTime / sec

k = 9.0 x 10-3 s-1

k = 9.6 x 10-3 s-1

0 20 40 60 800.00

Time / min

k = 6.9 x 10-4 s-1

k = 6.7 x 10-4 s-1pH = 7.4

pH = 7.4

300 400 500 6000.00

Wavelength / nm

Photoswitched Protonated (A2) Form

488 nm

405 nm

pH = 5.0

300 400 500 6000.00

Wavelength / nm

0 20 40 60 80 1000.0

2] / 1

Time / min

k = 5.6 x 10-4 s-1

[CB] /

k = 5.1 x 10-4 s-1

0 300 600 900 12000.0

2] / 1

Time / sec

k = 9.1 x 10-3 s-1

[CB] /

k = 1.0 x 10-2 s-1

Scheme of the Photoswitching

488 nm

405 nm

Photoswitched protonated form Non-fluorescent

intermediate

Fluorescent deprotonated form

= 3.2 ×10-4

= 0.37

3.6 ns

Protonated form

New trends in GFP-research

• Diffraction-unlimited microscopy in far field

Hell, S. W. Curr. Opin. Neurobiol. 2004, 14, 599.

New probes for fluorescence

Emission versus excitation spectrum

- Emission spectrum or fluorescence spectrum: one excites at one wavelength and scan the emission- monochromator.- Excitation spectrum : one fixes the emission monochromator at one wavelength and scans the excitation monochromator.- At low concentrations excitation spectra and emission spectra should be the same. Differences point to aggregation or other processes (see energy tranfer).

lightsource

excitation-monochro-mator

emission-monochro-mator

sampleel

detector

Excitation Sources

LampsXenonXenon/Mercury

LasersArgon Ion (Ar)Krypton (Kr)Helium Neon (He-Ne)Helium Cadmium (He-Cd)Krypton-Argon (Kr-Ar)

Arc Lamp Excitation SpectraIr

Xe Lamp

Hg Lamp

Ethidium

cis-Parinaric acid

Texas Red

PE-TR Conj.

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines

Definitions for fluorescence

M+ha 1M*

M+hfl (fluorescence kf)

3M* (intersystem crossing kISC)

M (internal conversion kIC)

(Products (dissociation kP ))

Bimolec. processes (kBM)

kBM =kQ[Q]

Characteristic timesAbsorption : 10-15 s

Vibrational relaxation : 10-12 10-10 sLifetime of S1 : 10-10 10-7 s

Intersystem crossing : 10-10 10-8 sInternal conversion : 10-11-10-9 s

Lifetime T1 : 10-6 – 1 s

• Extinction Coefficient

– refers to a single wavelength (usually the absorption maximum)

• Quantum Yield

– Qf is a measure of the integrated photon emission over

the fluorophore spectral band

Parameters

• quantum yield fl

/ (kfl+k

speciesexcitedofnumbertotal

cefluorescenviadecayingspeciesofnumber

radiative lifetime 0 = 1/ k •fl

• decay time fl

= 1/ ( kfl

+ kISC

Lifetime & decay time

Parameters•Transition dipole moment : direction of movement of electrons

Photobleaching• Defined as the irreversible destruction of an excited

fluorophore (discussed in later lecture)• Methods for countering photobleaching (see

microscopy)– Scan for shorter times

– Use high magnification, high NA objective

– Use wide emission filters

– Reduce excitation intensity

– Use “antifade” reagents (not compatible with viable cells)

• anisotropy r = (III - I)/ (III +2I)

• polarisation P = (III - I)/ (III +I)

Definitions for fluorescence

Principle of photoselection : using polarized excitation light mainly molecules excited that havea transition dipole parallel to the excitation light.

As a result, the fluorescence is also polarized, unless processes occur that ‘destroy’ the polarization

Processes can be : rotation of the molecule, energy transfer…

Relation between P and r =

In ensemble measurements r is most frequently used.

In absence of depolarization processes the fundamental of limiting anisotropy value r0 has a value between 0.4 and -0.2 depending on the angle between excitation and emission transition dipole.

Decay time of a fluorophore

SAMPLE

Excitation( pulse)

d[1M*]/dt = - (kfl+kISC+kIC+kQ[Q]) [1M*]

fl = 1/(kfl+kIC + kISC+kQ[Q])

[1M*] = [1M*]0exp(-t/ fl )

Fluo. response functionIfl(t) (1/fl )exp(-t/fl )

Solving the differential equation

Time resolved fluorescence : excitation of the sample with a pulse that is shorterthen the decay time of the fluorophore, typically 5 ns.

Time resolved fluorecence

P u l s e ds o u r c e

S a m p l e

T r i g g e r( s y n c )

D e l a y

0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

s t a r t s t o p

Basic principle of the TCSPC experiment

Period 1

Period 2

Period 3

Period 4

Period N

Result after many photons

Basic principle of the TCSPC experiment

CFD TAC

start stop reset

TAC time

Va fVa

Excitation source: - flash lamps + monochromatic filters (ns pulses up to 10 kHz rep.) - mode-locked lasers (ps pulses up to 82 MHz rep.) - pulsed semiconductor diode lasers - synchrotron radiation (UV excitation)Optical components: - polarization accessories - collection lens system - monochromator

Detection system: - PMT, MCP or APD

Electronics: - Delay, CFD, TAC, Amplifier, MCA, PC.

Statistics

< xi>=<ni>q

Px(i) = (1/x!)[(<xi>)x exp(-<xi>)] < xi>=<ni>q

pulse S <ni> MCP

Large number of pulses for

one event

Single Photon Counting !

Decayhistogram

Yi= (1/fl ) SexcT t exp(-i t/fl)

Fit functions for the decays :

iT(t)= Ajexp(-t/ j); exponential model

iT(t) = Aexp(-t/ fl-2 (t/ fl)1/2); nonexponential model

1050-1300

240-335

720-1080

PP2530-630

360-500

MCP START

TCSPC experiment at K.U.Leuven

0 1000 2000 3000 40001

10000 28 ps; 4096 channels

time / ps0 5 10 15 20 25

time / ns

Measurements under ‘magic angle’ in order to avoid distortions by rotational diffusion (magic angle is 54.7 degrees for vertical polarization).

polarizer

Time-resolved emission spectroscopy (TRES)

• provide information on the evolution of kinetics in terms of intensity, time and spectral position• solvent relaxation around fluorophores, short-lived species, molecules having two or more fluorescing configurations with different decay times are processes that can be studied using TRES.

0 2 4 6 8 10 12 14 16 18 200

time / ns

Time-resolved fluorescence depolarization measurements

• information about the molecular reorientational motion in solution.

r (t)= (III(t) - I(t))/ (III(t) +2I(t))

IT(t) = III(t) +2I(t)

III(t)=exp(- t/ fl)(1+2r0 exp(- t/ ))

I(t)=exp(- t/ fl)(1-r0 exp(- t/ ))

r = r0exp(- t/ )

III(0)

III(t)

1. fl< r : fluorescence decays before anisotropy only r0 can be measured

2. fl> r or fl r : r0 and r can be measured.

j randttr 0)/exp()(kT

• intra and intermolecular excited state processes taking place from picosecond to

nanosecond time scale.•determination of rates of competitive de-excitation pathways.•reaction kinetics: proton/electron and energy transfer, excimer or exciplex

formation. •environmental effects: solvent relaxation, quenching of excited states,

conformational dynamics in proteins.

IIIIII zyxtot 2//

66.0)7.54(33.0)7.54( 22 SinandCos

Energy Transfer

Radiative

Non radiative-Dexter type- Forster type

Energy transfer

- Energy transfer is iso-energetic, followed by fast vibrational relaxation

- Excited state of acceptor should be lower than that of donor to have driving force

- Quantum yield of donor and decay time of donor decrease.

- Process can occur between singulet as well as triplet excited states.

- Two mechanisms (except for trivial mechanisms) : Dexter and Förster transfer

D* A D A*

Energy transfer

- Dexter transfer : exchange mechanism, distances between 0.5 and 1 nm, spin changes are

allowed. Overlap between donor fluorescence and acceptor absorption required.

D* A D A*

Energy transfer

- Förster transfer : long distance, upto 10 nm, dipole-dipole interaction, total spin maintained,

resonance energy transfer. Overlap between donor fluorescence and acceptor absorption

required. Due to strong distance dependence also called ‘molecular ruler’.

- Förster transfer between identical chromophores is called energy hopping and can go in

both directions.

E is called the efficiency of energy transfer

Fluorescence

Fluorescene (Forster) Resonance Energy TransferFRET

Wavelength

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

Energy transfer

E can be obtained from the fluorescence quantum yield in the presence (QDA) and absence

of the acceptor (QD) (and in a similar way from decay time in presence and absence of acceptor).

It can be shown that the rate constant for transfer equals:

kQandQ

D is the decay time of the donor in absence of the acceptor, R is the distance between donor

and acceptor and R0 is the Förster radius, the distance at witch half of the excitation energy

undergoes transfer while half is dissipated by all the other processes including emission.

AVD NnJQR 45260 128/)10(ln9000

J is the so called overlap integral between emission and absorption and is the orientation

factor (2/3 for random orientation).

Energy transfer

The overlap integral can be calculated as :

The orientation factor can be written as:

4)()( dfJ AD

222 )coscos2cossin(sin)coscos3(cos ADADADT or

Energy transfer

Forster type Energy Transfer(FRET)

• Effective between 10-100 Å only

• Emission and excitation spectrum must significantly overlap

• Donor transfers non-radiatively to the acceptor

• PE-Texas Red™

• Carboxyfluorescein-Sulforhodamine B

Electron transfer

Intermolecular Electron transfer always occurs via collision and requires diffusion

(O2 will diffuse 7 nm in 10 ns in aqueous solution)

maximum rate constant for bimolecular reaction is in the order of 4x1010

D* A D A*Radical Ionpair

Excited donor is a better donor, excited acceptor is a better acceptor

Markus theory for e-transfer : theory that describes how the rate constant of electron transfer depends on parameters such as orientation,ΔG, solvent reorganization, distance….

FVhkET224

TkGTkVhk bbET 4exp)4(4 22122

)(exp0 tRktk ETET

2.1,10 1120 skET

Kinetics of quenching

The case of bimolecular quenching (stationairy)

K is Stern-Volmer constant in l.mol-1

111 SQkkISQkkkkkI

SdqdABSqpiscicfABS

kd is the rate constant for deactivation without quenching

Qkkkkk

qpiscicf

piscicf

qpiscicf

Qkkkkk

QkQK qf

of 011

Stern-Volmer equation

The case of bimolecular quenching (time resolved)

111 SQkkISQkkkkkI

SdqdABSqpiscicfABS

tStQkk

tQkkkkktStS

qpiscicf

exp)0(exp)0(

exp)0()(

QkkQkkkkk qdqpiscicf

QkQK q0

The case of intramolecular quenching

Solving the equation leads to

111 SkkISkkkkkI

SdqdABSqpiscicfABS

piscicf

qpiscicf

qdqpiscicf kkkkkkk

Examples

Fluorescence polarization

Anisotropy to study micro-viscosity in membranes and aggregation

Energy transfer

Distance determination form the extend of transfer

Energy transfer

R0 = 5 nm

Photosynthesis

Humans, animals, fungi, bacteria live by degrading molecules provided by other organisms…. Life on earth obviously could not continue indefinitely in this manner without an independent mechanism for synthesizing complex molecules from simple ones: the energy provided in this mechanism comes from the sun and is captured in the process of photosynthesis.

Plants and other photosynthetic organisms fixe 1011 tons/year of carbon in organic compounds (carbohydrate molecules, noted (CH2O)) from CO2. But globally, the consumption is higher than the synthesis…. So, what will happen?

CO2 + H2O + light (CH2O) + O2

Important to understand the photosynthesis and how our activities affect it!

Note: 1/3 of the fixed C is done by microorganisms in the oceans. Some bacteria also participate to the photosynthesis.

Equilibrium constant: K= 10-496

huge thermodynamic gradient!

Porphyrin ring

Chlorophyll structure

c.f. TZ 7.5

The First Step: absorption of light

• In addition to chlorophyll, plants contain several pigments that absorb light

• The accessory pigments have antioxidant functions as well

EXCITATION

light & heat

3 POSSIBLE DECAY PATHWAYS

excited pigment molecule

1. fluorescence2. resonance

energy transfer3. successive resonance energy

transfers

neighboring pigment molecule e- donor

e- acceptor

+ - + -

After Alberts Fig. 14-47

Energy transfer after light absorption

Chlorophyll

Pigment moleculesResonance transfer of light energy

Electron acceptor“Special pair” of chl a molecules

Carotenoid or other pigment

Note: for bacteria, the antenna systems are called LH-I and LH-II. They have this characteristic hollow cylinder shape. LH-I has a reaction center (RC) within this cylinder. LH-II has 9 bacteriochlorophylls outside the cylinder (to take the light) and 18 within the cylinder (to transfer the energy).

32 bacteriochlorophylls

18 + 9 bacteriochlorophylls

Events at the PS II reaction center

c.f. TZ 7.24

Photosynthesis and aerobic respiration complete a cycle

Energy hopping

Energy transfer in multichromoporic systems key-process in photosynthesis.

The energy transfer process influenced by :- extend of coupling between the chromophores.

- disorder (slow and fast fluctuations of the surrounding proteins )...

Why investigate multichromophoric systems?Why investigate multichromophoric systems?

Energy hopping

400 450 500 550 600 650 700 7500.0

Wavelength /nm

g0em g1r4em g0abs g1r4abs

Energy hopping

Compound r0 1 (ns) 2 (ns) 1

2 2/r0 (%) drot (Å)a dFRET (Å)b

I 0.38 0.95 - 0.38 - 0 23.3 -

II 0.31 1.1 0.20 0.16 0.15 48 24.3 26.5

III 0.28 1.2 0.13 0.10 0.18 63 25.3 26.1

IV 0.24 1.3 0.11 0.08 0.16 66 25.9 26.5

DFRETFRET k

khopp khopp

Energy hopping

Compound r0 1 (ns) 2 (ns) 1

2 2/r0 (%) drot (Å)a dFRET (Å)b

G1R1_p 0.34 1.4 0.34 - 0 26.18

G1R3_p 0.31 1.6 0.07 0.09 0.22 71 27.66 27.25

G1R4_p 0.34 1.96 0.05 0.07 0.27 79 29.29 27.06

khopp khopp = 4.6ns-1

Energy transfer

Combination

Energy transfer

Fluorescence decay analysis

Cameleon protein YC3.1

• Fluorescent indicators for measuring Ca2+ concentration.

- Energy donor : ECFP

ECFP EYFPCaM M13

440 nm 475 nm

EYFP440 nm

530 nm

- Energy acceptor : EYFP

- Linker : calmodulin (CaM)+

calmodulin-binding peptide M13 (myosin light chain kinase)

Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer between the flanking GFPs.

+4Ca2+

-4Ca2+

Definitions

• FRET: the excited donor transfers its energy to the acceptor via a dipole-dipole interaction.

• Requirements : - emission spectrum of donor and acceptor must overlap. - transition dipole moments of donor and acceptor must be sufficiently aligned. - distance between donor and acceptor must be such that probability of transfer is high.

• FRET can be detected by : - a decrease in donor decay time - a decrease in donor fluorescence intensity - an increase in acceptor

fluorescence intensity - a change in fluorescence

polarization - growing in component in acceptor decay

Absorption and emission spectra of EYFP

f (400 nm excitaiton) = 0.02

514 nm : deprotonated form.

f (500 nm excitaiton) = 0.61

400 nm : protonated form.

- Absorption spectrum

- Emission spectrum

528 nm : deprotonated form.

ESPT = 0.03

350 400 450 500 550 600 650

excitation spectrum absorption spectrum emission spectrum

Wavelength / nm

Excited-state photophysics of EYFP

560 nm 1 3.4

440 nm 0.9 0.006 0.1 0.06

a1 1 (ns) a2 2 (ns)

560 nm 1 3.4

excitation detection

400 nm

488 nm

20 (a)

det = 440 nm

det = 560 nm

0 1 2 3 4 5 6 7-5

time / ns

0 1 2 3 4 5 6 7-5

time / ns

450 500 550 6000

5.0-6.0 ns4.0-5.0 ns

3.0-4.0 ns2.0-3.0 ns

1.5-2.0 ns1.0-1.5 ns

0.8-1.0 ns0.6-0.8 ns

0.45-0.55 ns0.35-0.45 ns

0.25-0.35 ns0.15-0.25 ns0.1-0.15 ns

0.05-0.1 ns0-0.05 ns

Wavelength / nm

Excited-state photophysics of EYFP

ESPT60 ps, = 0.03

3.4 ns

A1* A2*

A1 A2 I B

- The A2* form having a conformation that allows ESPT, will relax to the I* state within 60 ps.

- The A1* form will decay radiatively to its corresponding ground state, its fluorescence being quenched down to 6 ps by a non-radiative process.

Photophysics of ECFP

0.01 0.24 0.10 1.0 0.89 3.2

a2 2 (ns) a3 3 (ns)a1 1 (ns)

300 400 500 600

Absorption spectrum Emission spectrum

Wavelength / nm

det = 480 nm

0 1 2 3 4 5 6 7-5

time / ns

ECFP and EYFP as an energy transfer pair

- The strong overlap of the emission spectrum of ECFP with the absorption spectrum of EYFP.

Although displaying complicated photophysics, ECFP and EYFP still can be used to construct an energy transfer pair.

- The relative high quantum yield of fluorescence of ECFP (f = 0.4).

- The mono-exponential decaying of fluorescence of EYFP when excited at the deprotonated band.

450 500 550 600 650 7000.0

Wavelength / nm

Emission spectra of YC3.1

ECFP EYFP

+4Ca2+ -4Ca2+

ID : the integrated fluorescence intensity of the donor

IA : the integrated fluorescence intensity of the acceptor

D : the fluorescence quantum yield of the donor

A : the fluorescence quantum yield of the acceptor

DA : the fluorescence quantum yield of the donor in the presence of acceptor

Ca2+-binding YC3.1 E = 0.29

Ca2+-free YC3.1 E = 0.16

The distance between ECFP and EYFP

R0 : the critical transfer distance

R : the distance between the donor and the acceptor

kET : the rate constant of energy transfer

kf : the rate constant of donor in the absence of acceptor

DA : the fluorescence quantum yield of the donor in the presence of acceptor

10ln9000 f

2 : orientation factor

n : the refractive index of the solvent

NA : Avogadro’s number

f() : the fluorescence spectrum of the donor normalized on the wavenumber scale

() : the molar extinction coefficient of the acceptor at that wavenomber

Ca2+-binding YC3.1 R = 57 Å

Ca2+-free YC3.1 R = 65 Å

The distance between ECFP and EYFP

ECFPEYFP

ECFP EYFP

Å24 Å

• Ca2+-binding YC3.1 R = 57 Å

• Ca2+-free YC3.1 R = 65 Å

>120 Å

The estimated R value is consistent with the proposed structure.

- Even for assuming the perfectly oriented transition dipole moment (2 = 4), the efficiency of the energy transfer is estimated to be E = 0.027 if the protein adopt the most extended conformation (R = 120 Å).

Relatively compact conformation of the protein construct, even in the Ca2+-free condition.

Johan Hofkens Laboratory of Photochemistry and Spectroscopy Katholieke Universiteit Leuven - Belgium...

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