Spectrally Silent Transformation
in the Photolysis of Octopus
Rhodopsin
Nishioku 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
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
• 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
• Based on
diffraction
pattern/interfer
ence pattern
http://www.ceravolo.com/images/sphere_hindle.jpg
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
http://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
bioworld 2010
http://www.bio-world.com/images/sepharose.jpg
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
• 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
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