SMS Photo Physics Herten - Events

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 / ms

(2')

/ 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