Single molecules& Photo-physics

Dirk-Peter HertenHeidelberg University

EMBO Course: F-Techniques

Heidelberg, 23. -27.9.2009

Molecules

OHO OH+

O

OH

OHO OH+

O

OH

Models

OHO OH+

O

OH

Human models

Modeling

IndividualAverageIndividual

IndividualModel

Ensemble=

AveragededuceModel

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

Dilute & Select

Mechanical selection:Near-field techniques: NSOM, AFM(Surfaces)

Spectral selectionSolid-state techniques(Glases)

Spatial selection:Far-field techniques: SMFS, F-techniques …

Laser

Detektor(APD)

Laser

CCD

Spatial selectionConfocal fluorescence microscopy:Diffraction limited excitation/detection (~ 1fl)

Total-internal reflection fluorescence microscopy (TIRFM):Evanescent wave (~ 100 nm)

Reject background

What does a single molecule look like?

This is a single molecule!

Enzymatic catalysis

E +S E +PK M k ca t

E S

• 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

Location Conformation

Constitution Redox-state

e-

+

Single-molecule fluorescence spectroscopy

Objective:Connect molecular states to changes in fluorescence emission.

Photo-physics / Photo-chemistry

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)

Dye

HOMO

LUMO

Reducing reagent

Energy 1. Excitation2. Reduction (ET1)3. Recombination (ET2)

short range effect (contact pair)

O

N

N+

N

CH3

O

OH

NH

NNH

N

O

NH2

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 A

Similar energy levels

Single pair FRET

**

*

DA

AFRET II

IE

g+=

0 5 10 15 200

20

40

60

80

time / s

coun

trat

e / k

Hz

I – intensityI* – background corrected intensityγ – crosstalk correction

• Solution (confocal microscope):• Limited by diffusion (1 – 2 ms)

• Immobilization:• time-resolved studies can resolve

(dynamic) heterogeneities and kinetics.

• limited by photo-bleaching.

time

inte

nsity IA

ID

Zero FRET efficiency

AA

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

700 800 900 1000 1100 1200 1300 1400 15000

10

20

30

40

50

60

0

10

20

30

40

50

60

phot

on c

ount

s pe

r m

s

time / ms

0,0

0,2

0,4

0,6

0,8

1,0

0,0

0,2

0,4

0,6

0,8

1,0

11-06-02 b64bisCy5-g-TMR @ ATP synthesis

prox

imity

fact

or

5200 5300 5400 5500 5600 5700 5800 5900 60000

10

20

30

40

0

10

20

30

40

10-06-02 b64bisCy5-g-TMR @ ATP hydrolysis

phot

on c

ount

s pe

r m

s

time / ms

0,0

0,2

0,4

0,6

0,8

1,0

0,0

0,2

0,4

0,6

0,8

1,0

prox

imity

fact

or

Hydrolysis of ATP: High – Medium – LowSynthesis of ATP: Low – Medium - High

Photo-physics is key to SMFS

• Förster Resonance Energy Transfer (FRET)distance dependence: 2 – 8 nm

• Photo-induced Electron Transfer (PET) distance dependence: < 1nm

• Charge-transfer (MO interaction): direct / transfer

• Changes in the chromophore: direct

• …

Location Conformation

Constitution Redox-state

- e-

+ e-

+.

Combining photo-physical processes

ATTO 520

Cy5

Double stranded DNA:

Stiff (persistence length of ~ 50 nm)

Defined distances (molecular ruler)

Established labeling procedures

Ideal scaffold to test photo-physical reactions

Kumbakhar et al., ChemPhysChem 2009

Balancing FRET and ET

Proximity / EFRET

EET

FRET/ET ET FRET

1 2 3 4 5 6 7 8

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.

spFRET experiments

AA

FRET Donor-only

Acceptor bleaching

Donor bleaching**

*

DA

AFRET II

IE

g+=

FRET efficiency distributions

Ensemble: 2 populations, (FRET & ET); Single-molecule: 1 population (FRET)

Fluorescence fluctuations

0 3 6 9 12 15 180

10

20

30

0 3 6 9 12 15 18 21 240

10

20

30

40

B

Cou

nt R

ate,

kH

z

C

Time (second)

FRET-only

FRET-ET

Fluorescence fluctuations:- After acceptor bleaching- Only in presence of guanine

Fluctuation kinetics

X

1, 2, 3 2’, 3’(mismatches)

DNA breathing

The longer the -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, IndiaHaridas PalManoj Kumbhakar

Alex KielKostas LymperopoulosDaniel SiegbergHaisen TaTanja ErhardDaniel BarzanChristina SpassovaJessica BalboMichael SchweringAnne SeefeldAnton KurzArina Rybina

EXC 81

Thank you!