1. Optical and computational studies of membrane protein structure Mikhail Proskurin, Štěpán Timr, Dzmitriy Turavets 2011/07/28

2. Fluorescent proteins in living cells Horizontal polarizationVertical polarization

3. Horizontal polarizationVertical polarization Fluorescent proteins in living cells

4. Horizontal + vertical polarization Dichroic ratio: F h / F v

5. Two-photon transition moment TM TM orientation ?

6. Measuring fluorescence of GFP crystals Excitation light polarization rotated 0-180°

7. F [a.u.] Polarization angle [deg] F [a.u.] Two-photon excitation 800 nmOne-photon excitation 405 nm Fluorescence of a GFP crystal

8. F [a.u.] Polarization angle [deg] F [a.u.] Two-photon excitation 800 nmOne-photon excitation 405 nm Fluorescence of a GFP crystal Mathematical processing → transition moment orientation

9. GFP two-photon transition moment φ φ = 5.3˚±2.0˚ (mean±2SEM)

10. Horizontal + vertical polarization Dichroic ratio: F h / F v

11. Transition moment orientation with respect to cell membrane Searching forα 0, σ Transition moment of a fluorescent protein attached to cell membrane

12. LD measurement

13. Fluorescence anisotropy measurement

14. Theoretical models log 2 (F h / F v ) prediction for vertically oriented membrane σ [rad] α 0 [rad] Linear dichroismFluorescence anisotropy

15. The dlGFP construct G. Miesenboeck & al., 2004 Side viewFront view

16. Preparing the dlGFP plasmid

17. Transfecting mammalian cells with dlGFP

18. Fluorescence of dlGFP-expressing cells Horizontal polarization of excitation light Vertical polarization of excitation light

19. Horizontal polarization of excitation light colored red Vertical polarization of excitation light colored green Fluorescence of dlGFP-expressing cells

20. Vertical polarization of excitation light colored green, horizontal polarization of excitation light colored red

21. Optical properties of dlGFP in living cells Linear dichroismFluorescence anisotropy

22. Matching dlGFP optical properties with tilt angle  0, distribution width  Linear dichroismFluorescence anisotropy α 0 [rad] σ [rad]S Least square fitting – sum of squared residuals (S) of log 2 (F h / F v ) depending on α 0 and σ

23. Linear dichroismFluorescence anisotropy α 0 [rad] σ [rad]S Least square fitting – sum of squared residuals (S) of log 2 (F h / F v ) depending on α 0 and σ Matching dlGFP optical properties with tilt angle  0, distribution width 

24. Conclusions  In principle, the method works  The mathematical model needs to be improved  Applications include studies of membrane protein structure and function and design of probes of molecular processes in living cells

25. Acknowledgments Josef Lazar Karolína Tošnerová Alexey Bondar UFB UNSB