Post on 12-Jan-2016
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
Overview of electromagnetic radiation
Absorption : electronic transition(s) in a molecule
Orbitals, molecular orbitals
Simplified Jablonski Diagram
S0
S’
1E
n er g
yS1
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
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
1 . 0
2 . 0
3 . 0
4 . 0
5 . 0
( x )
S 0
S 1
Q - Q e ( i n Å )
E ( i n e V )
24000 22320 20640 18960 172800E+0
2E+5
4E+5
6E+5
8E+5
1E+6 00
030201
04
Intensity a.u.
Wavenumber cm
04
Characteristics of stationary molecular fluorescence
- 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
1.0
2.0
3.0
4.0
5.0
( x )
S 0
S 1
r ( i n Å )
E ( i n e V )
24000 22320 20640 18960 172800E+0
5E+3
1E+4
1,5E+4
2E+4
2,5E+4
3E+4
3,5E+4
4E+4
0 0
0 3
0 2
0 1
0 4
Intensity a.u.
Wavenumber cm-1
Characteristics of stationary molecular fluorescence
- 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
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0
0,5
1
1,5
2
2,5
Wavelength (nm)
Absorbance Relative Intensity (a.u.)
0-0
0-0
0-1
0-2
0-10-2
0-3
0-3
G° = 24480 cm-1
s = 640 cm-1
h = 1560 cm-1
;
= 1290 cm-1
Characteristics of stationary molecular fluorescence
- Repulsive S1 PES results in a broad unstructured spectrum.- Maximum given by the AB line.- Symmetric (Gaussian) absorption band.
Characteristics of stationary molecular fluorescence
- 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.
S1
h-Mh-EEele
k
2,5 3 3,5 4 4,50
1
2
3
4
5
Distance (Å)
Energy (eV)
Erep
S0
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
Flu
ores
cnec
e In
tens
ity
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
HO
NN
O
N
OH
NH N
H2N
NH3+
NHNH2
O
O
HO
Serine65
Arginine96
Glutamine94
Tyrosine66
Glu222
Glycine67
Phenylalanine64
Histidine148
Other Probes of Interest
Other Probes of Interest
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 :
Other Probes of Interest
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.
On
Off
Inte
nsi
ty
488 nm
405 nm
Time
Steady-State Spectra of Dronpa
pH = 7.4 pH = 5.0
O
NN
O
NN
O
OH
• 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
Flu
ores
cenc
e In
tens
ity
Abs
orba
nce
Wavelength / nm300 400 500 600
Flu
ores
cenc
e In
tens
ity
A
bsor
banc
e
Wavelength / nm
300 400 500 6000.00
0.05
0.10
0.15
Abs
orba
nce
Wavelength / nm
Photoswitching of Dronpa at the Ensemble Level
488 nm
405 nm
300 400 500 6000.00
0.05
0.10
0.15
Abs
orba
nce
Wavelength / nm
0.00
0.05
0.10
0.15
0 300 600 900 12000.00
0.02
0.04
Ab
sorb
an
ceTime / sec
k = 9.0 x 10-3 s-1
Ab
ao
rba
nce
k = 9.6 x 10-3 s-1
0.00
0.05
0.10
0.15
0 20 40 60 800.00
0.02
0.04
Ab
sorb
an
ce
Time / min
k = 6.9 x 10-4 s-1
Ab
ao
rba
nce
k = 6.7 x 10-4 s-1pH = 7.4
pH = 7.4
300 400 500 6000.00
0.02
0.04
0.06
0.08
0.10
Ab
sorb
an
ce
Wavelength / nm
Photoswitched Protonated (A2) Form
488 nm
405 nm
pH = 5.0
pH = 5.0
300 400 500 6000.00
0.02
0.04
0.06
0.08
0.10
Ab
sorb
an
ce
Wavelength / nm
0.0
0.5
1.0
0 20 40 60 80 1000.0
0.5
1.0
1.5
[CA
2] / 1
0-6 M
Time / min
k = 5.6 x 10-4 s-1
[CB] /
10
-6 M
k = 5.1 x 10-4 s-1
0.0
0.5
1.0
0 300 600 900 12000.0
0.5
1.0
1.5
[CA
2] / 1
0-6 M
Time / sec
k = 9.1 x 10-3 s-1
[CB] /
10
-6 M
k = 1.0 x 10-2 s-1
Scheme of the Photoswitching
On
Off
Inte
nsi
ty
488 nm
405 nm
Time
Photoswitched protonated form Non-fluorescent
intermediate
S0
S1
Fluorescent deprotonated form
= 3.2 ×10-4
= 0.37
3.6 ns
Protonated form
14 ps
New trends in GFP-research
• Diffraction-unlimited microscopy in far field
Hell, S. W. Curr. Opin. Neurobiol. 2004, 14, 599.
New probes for fluorescence
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
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
rad
ian
ce a
t 0.
5 m
(m
W m
-2 n
m-1)
Xe Lamp
Hg Lamp
Ethidium
PE
cis-Parinaric acid
Texas Red
PE-TR Conj.
PI
FITC
600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines
Definitions for fluorescence
M+ha 1M*
S0
S2
S1
T1
T2
abso
rpti
on
fluo
resc
ence
phos
phor
esce
nce
ISC
ICVR
VR
M+hfl (fluorescence kf)
3M* (intersystem crossing kISC)
M (internal conversion kIC)
(Products (dissociation kP ))
Bimolec. processes (kBM)
ISC
IC
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
/ (kfl+k
ISC+k
IC+k
BM)
speciesexcitedofnumbertotal
cefluorescenviadecayingspeciesofnumber
radiative lifetime 0 = 1/ k •fl
• decay time fl
= 1/ ( kfl
+ kISC
+ kIC
+ kBM
)
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.
r
rP
2
3
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
P M T
T r i g g e r( s y n c )
D e l a y
F e m
M C A
T A C
A D C
C F D
0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0
0
5 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
C F D
P
P
Basic principle of the TCSPC experiment
Period 1
Period 2
Period 3
Period 4
Period N
Result after many photons
start
start
start
start
start
Basic principle of the TCSPC experiment
CFD TAC
start stop reset
Vol
tage
TAC time
Va fVa
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
start
xi
< xi>=<ni>q
Px(i) = (1/x!)[(<xi>)x exp(-<xi>)] < xi>=<ni>q
iti
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
OPO
1050-1300
Ti:Sa
PP32
240-335
720-1080
PP2530-630
Ar+
360-500
SSTOP
Mono
MCP START
FHG
TCSPC experiment at K.U.Leuven
0 1000 2000 3000 40001
10
100
1000
10000 28 ps; 4096 channels
coun
ts
time / ps0 5 10 15 20 25
100
1000
10000
co
un
ts
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
1000
2000
3000
4000
5000
con
ts
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/ )
I(0)
III(0)
I(t)
III(t)
r r
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
jj
j randttr 0)/exp()(kT
V
• 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*
S0S0
S1
S1
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*
S0
S0 S1
S1
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.
DIDT
T
kkk
kE
E is called the efficiency of energy transfer
Fluorescence
Fluorescene (Forster) Resonance Energy TransferFRET
Inte
nsi
ty
Wavelength
Absorbance
DONOR
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:
DID
DD
DIDT
DDA
D
DA
kk
kQand
kkk
kQandQ
QE
1
6
01
R
Rk
DT
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:
0
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
dt
SdqdABSqpiscicfABS
kd is the rate constant for deactivation without quenching
Qkk
k
Qkkkkk
k
qd
f
qpiscicf
ff
piscicf
q
piscicf
qpiscicf
f
of
kkkk
Qk
kkkk
Qkkkkk
1
QkQK qf
of 011
Stern-Volmer equation
Kinetics of quenching
The case of bimolecular quenching (time resolved)
with
111 SQkkISQkkkkkI
dt
SdqdABSqpiscicfABS
kd is the rate constant for deactivation without quenching
Stern-Volmer equation
t
tStQkk
tS
tQkkkkktStS
qd
qpiscicf
exp)0(exp)0(
exp)0()(
11
11
QkkQkkkkk qdqpiscicf
11
QkQK q0
0
11
Kinetics of quenching
The case of intramolecular quenching
Solving the equation leads to
111 SkkISkkkkkI
dt
SdqdABSqpiscicfABS
kd is the rate constant for deactivation without quenching
Stern-Volmer equation
piscicf
q
piscicf
qpiscicf
f
of
kkkk
k
kkkk
kkkkk
1
qdqpiscicf kkkkkkk
11 00
1
qkor
Examples
Fluorescence polarization
Anisotropy to study micro-viscosity in membranes and aggregation
Kinetics of quenching
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!
http
://w
ww
.life
.uiu
c.ed
u/go
vind
jee/
pape
rs/m
iles
tone
s.ht
ml
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
light
3 POSSIBLE DECAY PATHWAYS
e-
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
Rav
en F
ig 7
-13;
c.f
. T
Z 7
.7
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
NO
O
C
NO
O
C
NO
O
NO
O
C
NO
O
N
O
O
N
O
O
NO
O
C
NO
O
N
O
O
Energy hopping
400 450 500 550 600 650 700 7500.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
Flu
o. In
tens
ity
(no
rmal
ised
)
Abs
orb
ance
(no
rmal
ised
)
Wavelength /nm
g0em g1r4em g0abs g1r4abs
Energy hopping
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
T
Vrot
DFRETFRET k
Rd
.606
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
N O
OON
O
khopp khopp = 4.6ns-1
Energy transfer
N
O
O
N
O
O
N
O
O
O
N
O
O
O
O
Energy transfer
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
ECFP
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
Inte
nsi
ty
Ab
sorb
ance
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
0
4
8
12
16
20 (a)
det = 440 nm
kcn
ts
0
2
4
6
8
10(b)
det = 560 nm
0 1 2 3 4 5 6 7-5
0
5
time / ns
res
0 1 2 3 4 5 6 7-5
0
5
time / ns
450 500 550 6000
100
200
300
400
500
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
Co
un
ts
Wavelength / nm
Excited-state photophysics of EYFP
6 ps
ESPT60 ps, = 0.03
3.4 ns
A1* A2*
I*B*
A1 A2 I B
~ 48
0 nm
~ 40
0 nm
~ 40
0 nm
~ 51
4 nm
~ 51
4 nm
~ 52
8 nm
~ 48
0 nm
- 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
Inte
nsi
ty
Ab
sorb
ance
Wavelength / nm
0
2
4
6
8
10
det = 480 nm
kcn
ts
0 1 2 3 4 5 6 7-5
0
5
time / ns
res
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
0.2
0.4
0.6
0.8
1.0
1.2
Inte
nsi
ty
Wavelength / nm
Emission spectra of YC3.1
ECFP
EYFP
ECFP EYFP
+4Ca2+ -4Ca2+
DDAA
AA
D
DA
II
IE
1
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
fkk
k
RR
RE
ET
ET66
0
60
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
d
A
D 445
26
0 128
10ln9000 f
NnR
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
47 ×
32
× 30
Å24 Å
42 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.