Single molecules & Photo-physics

1. Single molecules& Photo-physics Dirk-Peter Herten Heidelberg University EMBO Course: F-TechniquesHeidelberg, 23. -27.9.2009

2. Molecules

3. Models

4. Human models

5. Modeling Individual Individual Model Average Individual Ensemble = Average Model deduce

6. 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

7. Single-molecule techniques Spatial selection: Far-field techniques: SMFS, F-techniques … Mechanical selection: Near-field techniques: NSOM, AFM (Surfaces) Spectral selection Solid-state techniques (Glases) Dilute & Select

8. Spatial selection Confocal fluorescence microscopy: Diffraction limited excitation/detection (~ 1fl) Total-internal reflection fluorescence microscopy (TIRFM): Evanescent wave (~ 100 nm) Reject background

9. What does a single molecule look like? This is a single molecule!

10. Enzymatic catalysis • 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 • …

11. Molecular transitions Single-molecule fluorescence spectroscopy Objective: Connect molecular states to changes in fluorescence emission.  Photo-physics / Photo-chemistry

12. Photo-physics & Photo-chemistry • Fluorescence Resonance Energy Transfer (FRET) • Photo-induced Electron Transfer (PET) • Redox Reactions (Oxidation / Reduction) • Protonation • Charge-Transfer Bands • ….

13. Photo-physics & Photo-chemistry • Fluorescence Resonance Energy Transfer (FRET) • Photo-induced Electron Transfer (PET) • Redox Reactions (Oxidation / Reduction) • Protonation • Charge-Transfer Bands • ….

14. Redox-state Lu et al. Science 282 (1998), 1877-1882

15. Photo-physics & Photo-chemistry • Fluorescence Resonance Energy Transfer (FRET) • Photo-induced Electron Transfer (PET) • Redox Reactions (Oxidation / Reduction) • Protonation • Charge-Transfer Bands • ….

16. Photo-induced electron transfer (PET) Reducing reagent Dye Energy • Excitation • Reduction (ET1) • Recombination (ET2) • short range effect (contact pair) LUMO HOMO

17. Folding the Tryptophan Cage Neuweiler et al., Angew. Chem. 2003

18. Photo-physics & Photo-chemistry • Fluorescence Resonance Energy Transfer (FRET) • Photo-induced Electron Transfer (PET) • Redox Reactions (Oxidation / Reduction) • Protonation • Charge-Transfer Bands • ….

19. 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.

20. FRET – Distance

21. FRET – Orientation κ2 – Orientationalparameter

22. FRET – Spectral Overlap D A  Similar energy levels

23. Single pair FRET IA • I – intensity • I* – background corrected intensity • γ – 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. ID

24. Zero FRET efficiency  Alternating Laser Excitation (ALEX)

25. 3 Example: F1F0-ATPase • 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

26. Directionality and kinetics of F1F0-ATPase rotation Hydrolysis of ATP: High – Medium – Low Synthesis of ATP: Low – Medium - High

27. 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 • …

28. Combining photo-physical processes ATTO 520 • Double stranded DNA: • Stiff (persistence length of ~ 50 nm) • Defined distances (molecular ruler) • Established labeling procedures • Ideal scaffold to test photo-physical reactions Cy5 Kumbakhar et al., ChemPhysChem 2009

29. Balancing FRET and ET EET Bulk data suggests competition between ET and FRET. Proximity / EFRET

30. spFRET experiments FRET Donor-only Acceptor bleaching Donor bleaching

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

32. Fluorescence fluctuations FRET-only • Fluorescence fluctuations: • After acceptor bleaching • Only in presence of guanine FRET-ET

33. Fluctuation kinetics X 1, 2, 3 2’, 3’ (mismatches)

34. DNA breathing The longer the p-stack the more probable ET interrupted by breathing or partial unzipping or by charge trapping.

35. 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

36. 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