spectrally silent transformation in the photolysis of octopus rhodopsin nishioku et al. (2001)...
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![Page 1: Spectrally Silent Transformation in the Photolysis of Octopus Rhodopsin Nishioku et al. (2001) Brandon Drescher BIO 603](https://reader034.vdocuments.site/reader034/viewer/2022042821/56649d7f5503460f94a62ecb/html5/thumbnails/1.jpg)
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
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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
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•Plasma membrane
•Cell body•Nucleus
•Synaptic region
Alberts et al. 2008
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Alberts et al. 2008
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http://cas.bellarmine.edu/tietjen/Laboratories/Eye07.gif
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Background – Fringe Spacing
• Spacing = element(s) present
http://www.ceravolo.com/images/sphere_hindle.jpg
• Based on diffraction pattern/interference pattern
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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
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Transient Grating Method
•Femptosecond pulse laser systemhttp://plasmonic.net/images/imageR9.jpg
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•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
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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
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Methods
•Con-A Sepharose▫Affinity purification▫Separation of
glycoproteins from cell surface
http://www.bio-world.com/images/sepharose.jpg
bioworld 2010
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Methods
•Laser flash photolysis system•TSP-1000 (Japanese) used for transient
absorbance during photochemical reactions
http://www.unisoku.com/img/img-LaserFlashPhotolysis.jpg
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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
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Scheme 1
•Chemical species•Lifetimes•“X”
Nishioku et al. (2001)
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Kinetics
….boooo
Nishioku et al. 2001
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Results•Figure 1:
▫light-induced transient absorption changes (543.5 nm)
▫No absorbance changes beyond 100 μs (mesorhodopsin → acid metarhodopsin)
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Results•Figure 2:
▫Kinetics of transient grating method▫840 nm probe wavelength
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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???
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Some Key Notes
•Kinetics affected by temperature▫Figure 4▫Expansion of microenvironment
•Page 2926▫“structure change is not the dynamics
around the chromophore”
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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)
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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
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No Questions?? Then off to Dinner!!
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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