Nishioku et al. (2001) - People Server at UNCW

Spectrally Silent Transformation
in the Photolysis of Octopus
Nishioku et al. (2001)
Brandon Drescher
BIO 603
• Rhodopsin ≈ 70%
photoreceptor membrane
• Lot of work on octopus
photoactivation mechanism
and rhodopsin in late 90s
▫ Nakagawa et al. (1997, 98, 99,
• Plasma membrane
• Cell body
• Nucleus
• Synaptic region
Alberts et al. 2008
Alberts et al. 2008
Background – Fringe Spacing
• Spacing =
• Based on
ence 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 system
• 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
• Sucrose floatation
▫ Isolation of membranes with sucrose gradient
▫ Sucrose monolaurate (SM 1200)
 Non-ionic detergent
• Con-A Sepharose
▫ Affinity purification
▫ Separation of
glycoproteins from cell
bioworld 2010
• Laser flash photolysis system
• TSP-1000 (Japanese) used for transient
absorbance during photochemical reactions
Hara et al. (1996)
• Enthalpy and volume change using the TG
Various alkanes as solvents
Beam splitter → focused through lens
Varying decay rates
Standard thermal grating signal for fringe spacing
Scheme 1
• Chemical species
• Lifetimes
• “X”
Nishioku et al. (2001)
Nishioku et al. 2001
• Figure 1:
▫ light-induced transient absorption changes (543.5
▫ No absorbance changes beyond 100 μs
(mesorhodopsin → acid metarhodopsin)
• 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
▫ 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
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)
• Spectral and kinetic
study into protein
• Use of many
compounds in
▫ Previous works
• Small time frames
▫ Figure 2
• Fairly hard to
understand if not
proficient in physical
• Required to go back
decades for clear
No Questions?? Then off to Dinner!!
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

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