Single molecules
& Photo-physics
Dirk-Peter Herten
Heidelberg University
EMBO Course: F-Techniques
Heidelberg, 23. -27.9.2009
Molecules
Models
Human models
Modeling
Individual
Individual
Individual
Model
Average
deduce
Model
Ensemble
=
Average
Why single molecules?
• Resolve molecular heterogeneities
– Static heterogeneties (Subpopulations)
– Dynamic heterogeneities (e.g. transitions
between different conformers)
– Resolve rare / hidden events
– Measure kinetics in thermodynamic equilibrium
• Ultimate limit of analytical sensitivity
Single-molecule techniques
Mechanical
selection:
Near-field
techniques:
NSOM, AFM
(Surfaces)
Spectral
selection
Solid-state
techniques
(Glases)
Dilute & Select
Spatial
selection:
Far-field
techniques:
SMFS, Ftechniques …
Spatial selection
Confocal fluorescence microscopy: Total-internal reflection
fluorescence microscopy (TIRFM):
Diffraction limited
excitation/detection (~ 1fl)
Evanescent wave (~ 100 nm)
Laser
Detektor
(APD)
Laser
CCD
Reject background
What does a single molecule
look like?
This is a single
molecule!
Enzymatic catalysis
KM
E+S
ES
kcat
E+P
• Substrate binding
 association
• Conformational change
• Allosteric interaction
• Co-enzyme binding
• Catalytic conversion
 ‘Chemical reaction’
• Series of elementary step
(protonation, cleavage, deprotonation,
substitution, oxidation, ….)
• Product dissociation
• …
Molecular transitions
Single-molecule
fluorescence
spectroscopy
Location
Conformation
e-
Constitution
Redox-state
+
Objective:
Connect molecular states
to changes in fluorescence
emission.
 Photo-physics / Photochemistry
Photo-physics & Photo-chemistry
• Fluorescence Resonance Energy
Transfer (FRET)
• Photo-induced Electron Transfer (PET)
• Redox Reactions (Oxidation /
Reduction)
• Protonation
• Charge-Transfer Bands
• ….
Photo-physics & Photo-chemistry
• Fluorescence Resonance Energy
Transfer (FRET)
• Photo-induced Electron Transfer (PET)
• Redox Reactions (Oxidation /
Reduction)
• Protonation
• Charge-Transfer Bands
• ….
Redox-state
Lu et al. Science 282 (1998), 1877-1882
Photo-physics & Photo-chemistry
• Fluorescence Resonance Energy
Transfer (FRET)
• Photo-induced Electron Transfer (PET)
• Redox Reactions (Oxidation /
Reduction)
• Protonation
• Charge-Transfer Bands
• ….
Photo-induced electron transfer
(PET)
OH
O
H3C
+
N
O
N
N
Dye
Energy
LUMO
HOMO
Reducing
reagent
1. Excitation
2. Reduction (ET1)
3. Recombination (ET2)
short range effect
(contact pair)
Folding the Tryptophan Cage
Neuweiler et al., Angew. Chem. 2003
Photo-physics & Photo-chemistry
• Fluorescence Resonance Energy
Transfer (FRET)
• Photo-induced Electron Transfer (PET)
• Redox Reactions (Oxidation /
Reduction)
• Protonation
• Charge-Transfer Bands
• ….
Fluorescence resonance energy
transfer (FRET)
• Non-radiative energy transfer
from an excited donor to an
acceptor dye.
• Strong distance dependence on
the range of 2 – 8 nm.
FRET – Distance
FRET – Orientation
κ2 – Orientational parameter
FRET – Spectral Overlap
D
 Similar energy levels
A
Single pair FRET
E FRET
I – intensity
I* – background corrected intensity
γ – crosstalk correction
IA
intensity
I *A
= *
I A + gI D*
• Solution (confocal microscope):
• Limited by diffusion (1 – 2 ms)
ID
time
countrate / kHz
80
60
40
20
0
0
5
10
time / s
15
20
• Immobilization:
• time-resolved studies can resolve
(dynamic) heterogeneities and
kinetics.
• limited by photo-bleaching.
Zero FRET efficiency
A
 Alternating Laser Excitation (ALEX)
Example: F1F0-ATPase
3
• Site-specific mutagenesis & labeling.
• Control of functionality.
• Reconstitution in vesicel membranes slow down diffusion 
extended observation time
Dietz et al., Nature Meth. Struct. Biol. 11 (2004), 135
Directionality and kinetics of
F1F0-ATPase rotation
Hydrolysis of ATP:
Synthesis of ATP:
High – Medium – Low
Low – Medium - High
11-06-02 b64bisCy5-g-TMR @ ATP synthesis
1,0
1,0
1,0
0,8
0,8
0,8
0,8
0,6
0,6
0,6
0,6
0,4
0,4
0,4
0,4
0,2
0,2
0,2
0,2
0,0
0,0
0,0
60
0,0
60
40
40
50
50
40
40
30
30
20
20
10
10
30
30
20
20
10
10
0
0
5200 5300 5400 5500 5600 5700 5800 5900 6000
time / ms
proximity factor
1,0
photon counts per ms
photon counts per ms
proximity factor
10-06-02 b64bisCy5-g-TMR @ ATP hydrolysis
0
700
800
900
0
1000 1100 1200 1300 1400 1500
time / ms
Photo-physics is key to SMFS
• Förster Resonance
Energy Transfer (FRET)
distance dependence:
2 – 8 nm
Location
Conformation
- e+ eConstitution
• Photo-induced Electron
Transfer (PET) distance
dependence: < 1nm
Redox-state
+.
• Charge-transfer (MO
interaction): direct /
transfer
• Changes in the
chromophore: direct
•…
Combining photo-physical
processes
ATTO 520
Double stranded DNA:
 Stiff (persistence length of ~ 50 nm)
 Defined distances (molecular ruler)
Cy5
 Established labeling procedures
 Ideal scaffold to test photo-physical
reactions
Kumbakhar et al., ChemPhysChem 2009
Balancing FRET and ET
FRET/ET
1
EET
ET
FRET
2
3
4
5
6
7
Φr
0.15
0.19
0.14
0.17
0.19
0.34
0.46
1.00
ED
0.80
0.51
0.10
-
0.91
0.73
0.51
-
EA
0.48
0.18
0.03
-
0.39
0.43
0.34
-
Bulk data suggests competition
between ET and FRET.
Proximity / EFRET
8
spFRET experiments
FRET
Donor-only
40
B
30
A
Count Rate, kHz
20
10
0
0
3
6
9
12
15
18
21
30
24
C
20
10
0
0
3
6
9
12
15
18
Time (second)
E FRET
I *A
= *
I A + gI D*
Acceptor
bleaching
Donor
bleaching
FRET efficiency distributions
Normalised number of occurence
5
1
6
2
7
3
0.4
0.6
0.8
1.0
FRET Efficiency (E)
Ensemble: 2 populations, (FRET & ET); Single-molecule: 1 population (FRET)
Fluorescence fluctuations
FRET-only
40
B
30
Count Rate, kHz
20
10
0
0
9
6
3
12
15
21
18
30
24
C
20
10
0
0
3
6
9
12
Time (second)
FRET-ET
15
18
Fluorescence fluctuations:
- After acceptor bleaching
- Only in presence of guanine
Fluctuation kinetics
A
<on> / ms
(2')
<off> / ms
B
6
3
(3')
2
(3)
X
(2)
(1)
21
(1)
18
15
12
(2)
9
(2')
(3')
(3)
6
3.5
4.0
4.5
R / nm
5.0
5.5
1, 2, 3
2’, 3’
(mismatches)
DNA breathing
The longer the p-stack the more probable ET
interrupted by breathing or partial unzipping or by
charge trapping.
Summary
• Photo-physics is key
– FRET: Distance, Spectral Overlap, Orientation
– PET: Short distance effect
– Redox Reaction
 Similar Energies / Redox potentials …
• Combining PET & FRET in dsDNA:
– SMFS reveals molecular heterogeneity
– fluorescence fluctuations indicate breathing of
dsDNA and electron transfer through π-stack
BARC, Mumbai, India
Haridas Pal
Manoj Kumbhakar
Alex Kiel
Kostas Lymperopoulos
Daniel Siegberg
Haisen Ta
Tanja Erhard
Daniel Barzan
Christina Spassova
Jessica Balbo
Michael Schwering
Anne Seefeld
Anton Kurz
Arina Rybina
Thank you!
EXC 81