Spectrally Silent Transformation in the Photolysis of Octopus Rhodopsin Nishioku et al. (2001)

1. Spectrally Silent Transformation in the Photolysis of Octopus RhodopsinNishioku et al. (2001) Brandon Drescher BIO 603 http://mrbarlow.files.wordpress.com/2007/08/octopus.jpg http://upload.wikimedia.org/wikipedia/commons/thumb/a/a1/All-trans-Retinal2.svg/439px-All-trans-Retinal2.svg.png

2. Background • Rhodopsin ≈ 70% photoreceptor membrane protein • Lot of work on octopus photoactivation mechanism and rhodopsin in late 90s • Nakagawa et al. (1997, 98, 99, 2001) Bovine Rhodopsin: http://www.ks.uiuc.edu/Services/Class/BIOPHYS490M/papers/Sakmar-minireview-2002.pdf http://www.ks.uiuc.edu/Research/rhodopsin/rh_in_membrane_bigger.gif

3. Plasma membrane • Cell body • Nucleus • Synaptic region Alberts et al. 2008

4. Alberts et al. 2008

5. http://cas.bellarmine.edu/tietjen/Laboratories/Eye07.gif

6. Background – Fringe Spacing • Based on diffraction pattern/interference pattern • Spacing = element(s) present http://www.ceravolo.com/images/sphere_hindle.jpg

7. Circular Dichroism • Spectroscopic measurements using left-handed polarized light vs. right-handed polarized light • Absorption differences based on structural (proteins) asymmetry • Protein structure stability • TG – alternative method for short time scales

8. Transient Grating Method • Femptosecond pulse laser system http://plasmonic.net/images/imageR9.jpg

9. TG method • Transient grating method • Spectral and kinetic-based analysis • Detection of protein dynamics with no optical absorption of the chromophore • Various contributions to sort out mathematically • Thermal grating, diffusion coefficients, refractive index change, density http://www.uni-potsdam.de/u/physik/Photonik/Gitter/grating1.png

10. Methods • Sucrose floatation • Isolation of membranes with sucrose gradient • Sucrose monolaurate (SM 1200) • Non-ionic detergent http://molecules.gnu-darwin.org/html/00150001_00175000/174829/174829.png

11. Methods • Con-A Sepharose • Affinity purification • Separation of glycoproteins from cell surface bioworld 2010 http://www.bio-world.com/images/sepharose.jpg

12. Methods • Laser flash photolysis system • TSP-1000 (Japanese) used for transient absorbance during photochemical reactions http://www.unisoku.com/img/img-LaserFlashPhotolysis.jpg

13. Hara et al. (1996) • Enthalpy and volume change using the TG method • Various alkanes as solvents • Beam splitter → focused through lens • Varying decay rates • Standard thermal grating signal for fringe spacing measurement

14. Scheme 1 • Chemical species • Lifetimes • “X” Nishioku et al. (2001)

15. Kinetics ….boooo Nishioku et al. 2001

16. Results • Figure 1: • light-induced transient absorption changes (543.5 nm) • No absorbance changes beyond 100 μs (mesorhodopsin→ acid metarhodopsin)

17. Results • Figure 2: • Kinetics of transient grating method • 840 nm probe wavelength

18. Some Key Notes • Probe wavelength affects refractive index change • Due to population grating • Thermal grating + volume grating intensities less sensitive • Shorter λ’s = enhanced intensities • Blue light illumination • 1800-1700 cm-1 band region • Change in carboxylic group of Asp-81???

19. Some Key Notes • Kinetics affected by temperature • Figure 4 • Expansion of microenvironment • Page 2926 • “structure change is not the dynamics around the chromophore”

20. Since 2001…. • Studies into G-protein activation + • Truncated octopus rhodopsin • Forward and reverse agonists (Ashida et al. 2004) • Len-free setup (Yamaguchi et al. 2003) • TG usage • Enthalpy measurementswith various solutions • High harmonic emissions (Mairesse et al. 2010)

21. Critique • Spectral and kinetic study into protein conformational change • Use of many compounds in methods • Previous works • Small time frames • Figure 2 • Fairly hard to understand if not proficient in physical chemistry • Required to go back decades for clear explanantion http://www.freewebs.com/andrej_gajic/Marine%20Biology/O%20Dofleini.jpg

22. No Questions?? Then off to Dinner!!

23. References • Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and P. Walter 2008. Molecular Biology of the Cell. Garland Science, New York 5th ed. Pgs. 917-919 • Ashida, A., Matsumoto, K., Ebrey, T.G. and M. Tsuda 2004. A purified agonist-activated G-protein coupled receptor: truncated octopus acid metarhodopsin. Zoological Science 21:245-250 • Hara, T., Hirota, N. and M. Terazima 1996. New application of the transient grating method to a photochemical reaction: the enthalpy, reaction volume change, and partial molar volume measurements. Journal of Physical Chemistry 100:10194-10200 • Mairesse, T., Dudovich, N., Zeidler, D., Spanner, M., Villeneuve, D.M. and P.B. Corkum 2010. Phase sensitivity of high harmonic transient grating spectroscopy. J. Phys. B: At. Mol. Opt. Physc. 43:065401 • Nishioku, Y., Nakagawa, M. Tsuda, M. and M. Terazima 2001. A spectrally silent transformation in the photolysis of octopus rhodopsin: a protein conformational change without any accompanying change of the chromophore’s absorption. Biophysical Journal 80:2922-2927 • Nakagawa, M., Kikkawa, S., Tominaga, K., Tsugi, N. and M. Tsuda 1998. A novel photointermediate of octopus rhodopsin activates its G-protein. FEBS Letters 436:259-262 • Ostrom, R.S. and X. Liu 2007. Detergent and detergent-free methods to define lipid rafts and caveolae. Methods in Molecular Biology 400:459-468 • Paulsen, R., Zinkler, D. and M. Delmelle 1983. Architecture and dynamics of microvillar photoreceptor membranes of a cephalopod. Experimental Eye Research 36:47-56 • Penelope the chipmunk – NGM ‘yourshot’ • Yamaguchi, M., Sawada, T., Katayama, K. and M. Fujinami 2003. Development of lens-free transient grating method and its application. Nippon KagakkaiKoenYokoshu 83:410