Spectrally Silent Transformation in the Photolysis of Octopus RhodopsinNishioku et al. (2001)Brandon DrescherBIO 603

http://mrbarlow.files.wordpress.com/2007/08/octopus.jpghttp://upload.wikimedia.org/wikipedia/commons/thumb/a/a1/All-trans-Retinal2.svg/439px-All-trans-Retina

l2.svg.png

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)

http://www.ks.uiuc.edu/Research/rhodopsin/rh_in_membrane_bigger.gif

Bovine Rhodopsin:http://www.ks.uiuc.edu/Services/Class/BIOPHYS490M/papers/Sakmar-minireview-2002.pdf

•Plasma membrane

•Cell body•Nucleus

•Synaptic region

Alberts et al. 2008

Alberts et al. 2008

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

Background – Fringe Spacing

• Spacing = element(s) present

http://www.ceravolo.com/images/sphere_hindle.jpg

• Based on diffraction pattern/interference pattern

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

Transient Grating Method

•Femptosecond pulse laser systemhttp://plasmonic.net/images/imageR9.jpg

•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

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

Methods

•Con-A Sepharose▫Affinity purification▫Separation of

glycoproteins from cell surface

http://www.bio-world.com/images/sepharose.jpg

bioworld 2010

Methods

•Laser flash photolysis system•TSP-1000 (Japanese) used for transient

absorbance during photochemical reactions

http://www.unisoku.com/img/img-LaserFlashPhotolysis.jpg

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

Scheme 1

•Chemical species•Lifetimes•“X”

Nishioku et al. (2001)

Kinetics

….boooo

Nishioku et al. 2001

Results•Figure 1:

▫light-induced transient absorption changes (543.5 nm)

▫No absorbance changes beyond 100 μs (mesorhodopsin → acid metarhodopsin)

Results•Figure 2:

▫Kinetics of transient grating method▫840 nm probe wavelength

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???

Some Key Notes

•Kinetics affected by temperature▫Figure 4▫Expansion of microenvironment

•Page 2926▫“structure change is not the dynamics

around the chromophore”

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 measurements with various

solutions▫High harmonic emissions (Mairesse et al. 2010)

Critique•Spectral and

kinetic study into protein conformational change

•Use of many compounds in methods▫Previous works

•Small time frames▫Figure 2

http://www.freewebs.com/andrej_gajic/Marine%20Biology/O%20Dofleini.jpg

• Fairly hard to understand if not proficient in physical chemistry

• Required to go back decades for clear explanantion

No Questions?? Then off to Dinner!!

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 Kagakkai Koen Yokoshu 83:410