3.4 lecture Flashcards
Aequorin
-luminescent protein
-calcium binding causes a
conformational change that results in emission of a blue light photon
Green Fluorescent Protein (GFP)
a fluorescent protein
-absorbs the photon of blue light from aequorin to become excited
-emits a photon of green light
What is aequorin’s chromophore?
-coelenterazine
-coordinated by Tyr184
How does aequorin bind to calcium?
3 EF hand domains bind to calcium
Luminescent proteins
Dissipate chemical/mechanical energy by emitting the energy as a photon
Fluorescent proteins
Absorbs photon/light energy to become excited and then emits a 2nd photon to dissipate the energy (GFP)
Excitation
Wavelengths absorbed
Emission
emits light
Which always has lower energy excitation or emission?
Emitted photons because some energy is lost
Green Fluorescent Protein (GFP) structure?
-tight11 beta barrel
-chromophore is at center if barrel
-hydrophobic beta barrel protects chromophore from ions and excess oxygen
Wha3 residues is GFP’s chromophore made of?
- Ser65
- Tyr66
- Gly67
How do GFP mutations change color?
These mutations alter the energy
input required for excitation and the
energy output released upon emission
DsRed
-fluorescent red protein
-tetramer
-prone to slow folding and misfolding
-33 amino acid substitution needed to make mRFP1 (first red protein)
mCherry
-optimized version of mRFP1
-more intense color
How is a calcium ion sensor made?
It is made by fusing mutant GFP to calmodulin
How is the intensity of the fluorescence emission manipulated?
-when Ca2+ binds to calmodulin the conformational change makes gap in the GFP barrel structure so fluorescence is no longer quenched and GFP fluoresces
What are fluorescent timer proteins and how do they work?
-track age of proteins in the cell
-when 1st expressed it is one color but then changes to another color due to chromophore oxidation
What is FRET
-Fluorescence Resonance Energy Transfer, which uses 1 fluorescent protein to excite another
-only happens if proper wavelength is used and close in proximity
What color is fluorescence when CFP is far from YFP?
Cyan fluorescence
-proteins do not interact
What color is fluorescence when CFP is close to YFP?
Yellow fluorescence
-proteins do interact
