AequorinAequorinThe Blue The Blue

Fluorescent ProteinFluorescent ProteinSarah McPeakSarah McPeakBiochemistry 310Biochemistry 310Dr. Dick LuraDr. Dick LuraDecember 7, 2004December 7, 2004

BioluminescenceBioluminescence The release of “cold light” from an The release of “cold light” from an

organism.organism.Caused by the oxidation of a Caused by the oxidation of a

substance within the organism.substance within the organism.Biological chemical reaction that Biological chemical reaction that

induces an excited state then induces an excited state then radiativly decays producing light.radiativly decays producing light. A A A*A*A + hvA + hv

Bioluminescent reaction of Bioluminescent reaction of Aequorin (overview)Aequorin (overview)

Chemical ReactionChemical Reaction

UsesUsesUsed as a marker in plants.Used as a marker in plants.

Studied for comparison to Studied for comparison to other photoproteinsother photoproteins

Used as a detector for CaUsed as a detector for Ca2+2+

Pre-crystal StructurePre-crystal Structure Used Circular Dichroism Spectra to Used Circular Dichroism Spectra to

look at portions of the secondary look at portions of the secondary structure and the effects point structure and the effects point mutations had on it.mutations had on it.

Concluded that position 71 and 84 Concluded that position 71 and 84 were likely candidate for the were likely candidate for the coelenterazine docking cite coelenterazine docking cite

CDCD

Indicates % of secondary structuresIndicates % of secondary structures

Crystal StructureCrystal Structure Small crystals formed using hanging Small crystals formed using hanging

drop vapor diffusiondrop vapor diffusion Found that there was actually two Found that there was actually two

different forms of the protein though different forms of the protein though they were very similarthey were very similar

Four EF-Hand domainsFour EF-Hand domainsTwo pairTwo pairEach is back to back connected by a beta Each is back to back connected by a beta

sheetsheet

Crystal StructureCrystal Structure Cups are hydrophobicCups are hydrophobic

Coelenterazine binds thereCoelenterazine binds there Peroxide on the Coelenterazine is Peroxide on the Coelenterazine is

stabilized by hydrogen bondingstabilized by hydrogen bondingTyr 184Tyr 184

The one EF-hand that does not accept The one EF-hand that does not accept a calcium may serve as a backbonea calcium may serve as a backbone

Explanation of Rnx From Explanation of Rnx From Crystal StructureCrystal Structure

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

Crystal StructureCrystal Structure

PropertiesProperties Gives off Blue light (466)Gives off Blue light (466) Heat ResistantHeat Resistant

Not when coupled physically with Not when coupled physically with GFPGFP

2.1 power relationship between [Ca2.1 power relationship between [Ca2+2+] ] and luminescent intensityand luminescent intensity10 fold increase = ~100 fold 10 fold increase = ~100 fold

increaseincrease

ProductionProduction Harvested from jellyfish Harvested from jellyfish Aequorea VictoriaAequorea Victoria

2 tons of jellyfish = 125 mg protein2 tons of jellyfish = 125 mg proteinEasily contaminated and can be harmful Easily contaminated and can be harmful

to the organismsto the organisms Clone using Clone using Escherichia coliEscherichia coli

After purified Charged with After purified Charged with coelenterazinecoelenterazine

Safe to useSafe to useIn vivo for small cellsIn vivo for small cellsLarger cells can be microinjected Larger cells can be microinjected

Aequorin in NatureAequorin in Nature

The luminescence from the The luminescence from the aequorin excites GFP causing the aequorin excites GFP causing the green glow of the jellyfishgreen glow of the jellyfish

Coelenterazine is a substrate that Coelenterazine is a substrate that is needed for the rnx to workis needed for the rnx to workThis is essential part of the jellyfish’s This is essential part of the jellyfish’s

dietdietMonterey bay aquarium study (MBA)Monterey bay aquarium study (MBA)

Monterey Bay AquariumMonterey Bay Aquarium

Used jelly fish that had no diet consumption Used jelly fish that had no diet consumption of coelenterazineof coelenterazine Upon stimulation no light foundUpon stimulation no light found Fluorescence microscopy indicated GFP was Fluorescence microscopy indicated GFP was

presentpresent Small amounts of jellyfish were soaked in the Small amounts of jellyfish were soaked in the

coelenterazine produced blue glowcoelenterazine produced blue glow Allows to consume other jellyfishAllows to consume other jellyfish

GlowedGlowed Given CoelenterazineGiven Coelenterazine

GlowedGlowed

CaCa2+2+ indicator indicator Able to monitor different portions of the Able to monitor different portions of the

cell with specific mutants of the aequorincell with specific mutants of the aequorin Used quite frequently in plant studiesUsed quite frequently in plant studies

Used to indicate stress factorsUsed to indicate stress factorsCalcium ion concentration increases Calcium ion concentration increases as stresses are addedas stresses are added

Used as a marker to observe the Used as a marker to observe the tobacco mosaic virus on tobacco tobacco mosaic virus on tobacco plantsplants

CaCa2+2+ in Plants in Plants Use Use

epifluorescent epifluorescent microscopes to microscopes to monitor the monitor the calcium calcium concentration.concentration.

ConsConsResponds Slowly to rapid Responds Slowly to rapid

change in Calciumchange in CalciumMgMg2+2+ effects luminescent out effects luminescent out

putputProduces low light outputProduces low light output

Need special instrumentationNeed special instrumentationi.e. CCD Camerai.e. CCD Camera

ProsPros Very low background because there is Very low background because there is

not excitation source beamnot excitation source beam High detection rangeHigh detection range Photo damage is not an issuePhoto damage is not an issue

Can be recharged with coelenterazineCan be recharged with coelenterazine Chance for future studies looks brightChance for future studies looks bright

Continually mutating to fit specific needsContinually mutating to fit specific needs

AcknowledgementsAcknowledgements

Michael RoachMichael Roach Dan McCauleyDan McCauley University of Virginia LibraryUniversity of Virginia Library Dr. Dick LuraDr. Dick Lura

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