1. Aequorin The Blue Fluorescent Protein Sarah McPeak Biochemistry 310 Dr. Dick Lura December 7, 2004

2. Bioluminescence  The release of “cold light” from an organism.  Caused by the oxidation of a substance within the organism.  Biological chemical reaction that induces an excited state then radiativly decays producing light.  A  A*  A + hv

3. Bioluminescent reaction of Aequorin (overview)

4. Chemical Reaction

5. Uses  Used as a marker in plants.  Studied for comparison to other photoproteins  Used as a detector for Ca 2+

6. Pre-crystal Structure  Used Circular Dichroism Spectra to look at portions of the secondary structure and the effects point mutations had on it.  Concluded that position 71 and 84 were likely candidate for the coelenterazine docking cite

7. CD Indicates % of secondary structures

8. Crystal Structure  Small crystals formed using hanging drop vapor diffusion  Found that there was actually two different forms of the protein though they were very similar  Four EF-Hand domains  Two pair  Each is back to back connected by a beta sheet

9. Crystal Structure  Cups are hydrophobic  Coelenterazine binds there  Peroxide on the Coelenterazine is stabilized by hydrogen bonding  Tyr 184  The one EF-hand that does not accept a calcium may serve as a backbone

10. Explanation of Rnx From Crystal Structure  “Displacement of the helices flanking the C- terminal loop would be expected to disrupt the hydrogen-bonding network of the loop, resulting in a relocation of the side chain of Tyr 184. This, in turn, would disrupt the hydrogen bounds to His 169 and the peroxide. No longer stabilized, the peroxide would be free to attack the adjacent carbonylic C3 to initiate the light-emitting reaction.”8

11. Crystal Structure

12. Properties  Gives off Blue light (466)  Heat Resistant  Not when coupled physically with GFP  2.1 power relationship between [Ca 2+ ] and luminescent intensity  10 fold increase = ~100 fold increase

13. Production  Harvested from jellyfish Aequorea Victoria  2 tons of jellyfish = 125 mg protein  Easily contaminated and can be harmful to the organisms  Clone using Escherichia coli  After purified Charged with coelenterazine  Safe to use  In vivo for small cells  Larger cells can be microinjected

14. Aequorin in Nature  The luminescence from the aequorin excites GFP causing the green glow of the jellyfish  Coelenterazine is a substrate that is needed for the rnx to work  This is essential part of the jellyfish’s diet  Monterey bay aquarium study (MBA)

15. Monterey Bay Aquarium  Used jelly fish that had no diet consumption of coelenterazine  Upon stimulation no light found  Fluorescence microscopy indicated GFP was present  Small amounts of jellyfish were soaked in the coelenterazine produced blue glow  Allows to consume other jellyfish  Glowed  Given Coelenterazine  Glowed

16. Ca 2+ indicator  Able to monitor different portions of the cell with specific mutants of the aequorin  Used quite frequently in plant studies  Used to indicate stress factors  Calcium ion concentration increases as stresses are added  Used as a marker to observe the tobacco mosaic virus on tobacco plants

17. Ca 2+ in Plants  Use epifluorescent microscopes to monitor the calcium concentration.

18. Cons  Responds Slowly to rapid change in Calcium  Mg 2+ effects luminescent out put  Produces low light output  Need special instrumentation  i.e. CCD Camera

19. Pros  Very low background because there is not excitation source beam  High detection range  Photo damage is not an issue  Can be recharged with coelenterazine  Chance for future studies looks bright  Continually mutating to fit specific needs

20. Acknowledgements  Michael Roach  Dan McCauley  University of Virginia Library  Dr. Dick Lura

21. Sources ► 1. Aequorin: A Bioluminescent Ca2+ Indicator. Molecular Probes. 30 Nov. 2004. ► 1. Aequorin: A Bioluminescent Ca2+ Indicator. Molecular Probes. 30 Nov. 2004. ► 2. Varkova, S. V. et. al. “Obelin from the Bioluminescent Marine Hydroid Obelia geniculata: Cloning, Expression, and Comparison of some Properties with Those of Other Ca2+ -Regulated Photoproteins.” Biochemistry. 41 (2002): 2227-2236. ► 3. Pichler, A., Prior, J. L., Piwnica-Worms, D. “Imaging Reversal of Multidrug Resistance in Living Mice with Bioluminescence: MDR1 P- glycoprotein Transports Coelenterazine.” PNSA. 101.6 (2004) 1702-1707. ► 4. Inouye, S. “Blue Fluorescent Protein from the Calcium-sensitive Photoprotein Aequorin is a Heat Resistant Enzyme, Catalyzing the Oxidation of Coelenterazine.” Federation of European Biochemical Societies. 557 (2004): 105-110. ► 5. Lu Deng, Svetlana V., et. al. “Crystal Structure of a Ca2+ -discharged Photoprotein.” The Journal of Biological Chemistry. 279.23 (2004) 33647-33652. ► 6. Vysotski, E. S., Lee, J. “Ca2+ -Regulated Photoproteins: Structural Insight into the Bioluminescence Mechanism. Accounts of chemical Research. 37.6 (2004): 405-415. ► 7. Creton, R., Dreiling, J. A., Jaffe, L. F. “Calcium Imaging with Chemiluminescence.” Microscopy Research and Technique. 46 (1999): 390- 397. ► 8. Head, J. F., Inouye, S., Teranishl, K., Shimomura, O. “The Crystal Structure of the Photoprotein aequorin at 2.3 A Resoulution.” Nature. 405 (2000): 372-376. ► 9. Lewis, J. C., Lopez-Moya, J.J. Daunert, S. “Bioluminescence and Secondary Structure Properties of Aequorin Mutants Produced for Site- Specific Conjugation and Immobilization.” Bioconjugate Chemistry. 11 (2000): 65-70. ► 10. Circular Dichroism Spectroscopy. Dec. 3 2004 ► 10. Circular Dichroism Spectroscopy. Dec. 3 2004 ► 11. Haddock, S. H. D., Rivers, T. J., Robinson, F. H. “Can Coelenterates Make Coelenterazine? Dietary Requirement for Luciferin in Cnidarian Bioluminescence.” PNSA. 98.20 (2001): 11148-11151. ► 12. Mithofer, A., Mazars, C. “Equorin-Based Measurements of Intracellular Ca2+ -Signatures in Plant Cells.” Biological Procedures Online. 4.1 (2002): 105-118. <www.biologicalprocedures.com/bpo/arts/1/40/ ► m40.pdf> ► 13. Diveki, A., Salanki, K., Balazs, E. “Limited Utility of Blue Fluorescent Protein (BFP) in Monitoring Plant Virus Movement.” Biochimie. 84 (2002): 997-1002. ► 14. Logan, D. C., Knight, M. R. “Mitochondrial and Cytosolic Calcium Dynamics are Differentially Regulated in Plants.” Plant Physiology. 133 (2003): 21-24.