1. Fluorescence Depolarization Martin Cole, Faraz Khan Physics 200 Professor Newman http://www.mi.infm.it/~biolab/tpe/tutor/fpa/anis2.html

2. Fluorescence Electrons are excited to higher energy states, jumping them to a higher energy orbital Electrons relax to give off heat (non- radiative) and photons (radiative) Electrons can also spin flip to form a triplet spin-parallel state

3. The Jablonski Diagram

4. Rates The rate of absorption is extremely fast, on the order of 10 -15 seconds Internal conversion from S 2 to S 1 takes more time, on the order of 10 -12 seconds, but is still very fast The emission process can take as long as 10 -8 seconds, still fast, but slower than the other two processes by quite a lot

5. Size and Time If a fluorescent group is oriented in a rigid manner, it emits light with polarity As the group spins, the polarity is reduced and becomes more random Large macromolecules spin slowly relative to emission rates, and produce largely polar photons Small molecules rotate in the time it takes to emit, and produce a more randomized spectrum of photons

6. Fluorescent Probes Three categories: ◦ Intrinsic: naturally occurring, includes NADH, FAD, tryptophan and tyrosine ◦ Intrinsic Analogs: residue replacement with a fluorescent and synthetic molecule ◦ Extrinsic: Probes added that bind to the target molecule to fluoresce, very common

7. Steady State Depolarization Consider a plane of polarized light, moving in direction x with electric vector in z direction We call I ║ the intensity of light polarized in the z direction and I ┴ the intensity of light polarized in the x direction We can determine anisotropy (lack of uniform directionality) and polarization my measuring the intensities

8. Polarization and Anisotropy A (anisotropy) = (I ║ - I ┴ ) / (I ║ + 2I ┴ ) P (polarization) = (I ║ - I ┴ ) / (I ║ + I ┴ ) If there were no polarization, I ║ = I ┴ and P and A become 0 For a perfectly rigid molecule, P max is ½ and A max is 2/5

9. Rigid Molecule P 0 = (3cos 2 ζ –1) / (cos 2 ζ +3) A 0 = (3cos 2 ζ –1) / 5 Where ζ is the angle between absorption and emission dipoles

10. Time-Resolved Fluorescence Depolarization Two main types: ◦ Decay of emission: measures fluorescence after excitation pulse to determine fluorescent lifetime of fluorophore ◦ Anisotropic decay: measures reorientation of emission dipole to give information of translational and rotational movement of molecule

11. Perrin Equation A 0 = A F / (1+ τ F / τ c ) ◦ τ F is lifetime of fluorophore ◦ τ c is the rotational correlation time If we find that τ c is much bigger than τ F, we find that A 0 = A F

12. Instrumentation Methods of obtaining time-resolved fluorescent data ◦ Harmonic response - measures emission from a sinusoidally modulated excitation ◦ Impulse-response – directly observes emission decay following a short excitation impulse  Uses titanium-sapphire lasers to produce extremely brief pulses (subpicosecond)

13. Anisotropy Measurements Two main instrument formats: ◦ T - faster method that measures both parallel and orthogonal to incoming polarized beam ◦ L - single emission channel is used, emission is detected at a right angle to the excitation beam from scattering Introduces the correlation factor G to the perpendicular component of the A and P equations described before

14. Axis Modulation We can flip the polarization of our excitation beam between horizontal and vertical For vertical excitation, we sum emitted intensities I VH and I VV to get that A V = I VH + I VV For horizontal excitation, we find that A H = 2I VH

15. Calculations From A v and A H, we can calculate the anisotropy A=(A v -A H ) / (A v + ½(A H )) This method of anisotropic determination does not require the G factor correction

16. Static Polarization Constant Illumination ◦ Use average Anisotropy equations 2 Hopkins, S., Sabido-David, C., Corrie, J., Irving, M., & Goldman, Y. (1998). Fluorescence Polarization Transiets from Rhodamine Isomers on Myosin Regulatory Light Chain in Skeletal Muscle Fibers. Biophysical Journal, 74, 3093-3110. 1

17. Hopkins et al Probe http://www.biochemj.org/bj/440/bj4400043add.htm

18. τ cor and Rotational Diffusion 3 http://www.youtube.com/watch?v=A_HyVm6UTM8 http://www.glycoforum.gr.jp/science/word/glycotechnology/GT-C06E.html Neyroz, P., Menna, C., Polverini, E., & Masotti, L. (1996). Intrinsic Fluorescence Properties and Structural Analysis of p13suc1 from Schizosaccharomyces pombe. Journal of Biological Chemistry, 271, 27249-27258.

19. Perrin Equation for Anisotropy Albani, J. (2010). Fluorescence properties of porcine odorant binding protein Trp 16 residue. Journal of Luminescence, 130 (11), 2166-2170. 4

20. Anisotropy Decay Schlosser, M., & Lochbrunner, S. (2006). Exciton Migration by Ultrafast Förster Transfer in Highly Doped Matrices. Journal of Physical Chemistry, 110, 6001-6009. 5

21. Ellipsoid Corrections Relation of Anisotropy with time can be expanded to three exponentials if macromolecules are viewed as ellipsoids http://science.yourdictionary.com/ellipsoid

22. Anisotropy and Molecular Weight Kay, L., Torchia, D., & Bax, A. (1989). Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. Biochemistry, 28 (8972). 6

23. Dependence on Lifetime Pope, A., Haupts, U., & Moore, K. (1999). Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice. Drug Discovery Today, 4 (8), 350-362. 7

24. Interesting Experiments 8 Whitson, K., Beechem, J., Beth, A., & Staros, J. (2004). Preparation and characterization of Alexa Fluor 594- labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor. Analytical Biochemistry, 324 (2), 227-236.

25. References 1 Hopkins, S., Sabido-David, C., Corrie, J., Irving, M., & Goldman, Y. (1998). Fluorescence Polarization Transiets from Rhodamine Isomers on Myosin Regulatory Light Chain in Skeletal Muscle Fibers. Biophysical Journal, 74, 3093-3110. 2 Serdyuk, I., Zaccai, N., & Zaccai, J. (2007). Methods in Molecular Biophysics: Structure, Dynamics, Function. Cambridge: Cambridge University Press. 3 Neyroz, P., Menna, C., Polverini, E., & Masotti, L. (1996). Intrinsic Fluorescence Properties and Structural Analysis of p13suc1 from Schizosaccharomyces pombe. Journal of Biological Chemistry, 271, 27249-27258. 4 Albani, J. (2010). Fluorescence properties of porcine odorant binding protein Trp 16 residue. Journal of Luminescence, 130 (11), 2166-2170. 5 Schlosser, M., & Lochbrunner, S. (2006). Exciton Migration by Ultrafast Förster Transfer in Highly Doped Matrices. Journal of Physical Chemistry, 110, 6001-6009 6 Kay, L., Torchia, D., & Bax, A. (1989). Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. Biochemistry, 28 (8972). 7 Pope, A., Haupts, U., & Moore, K. (1999). Homogeneous fluorescence readouts for miniaturized high- throughput screening: theory and practice. Drug Discovery Today, 4 (8), 350-362. 8 Whitson, K., Beechem, J., Beth, A., & Staros, J. (2004). Preparation and characterization of Alexa Fluor 594- labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor. Analytical Biochemistry, 324 (2), 227-236.