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Biochemistry > 3.4 lecture > Flashcards

3.4 lecture Flashcards

1
Q

Aequorin

A

-luminescent protein
-calcium binding causes a
conformational change that results in emission of a blue light photon

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2
Q

Green Fluorescent Protein (GFP)

A

a fluorescent protein
-absorbs the photon of blue light from aequorin to become excited
-emits a photon of green light

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3
Q

What is aequorin’s chromophore?

A

-coelenterazine
-coordinated by Tyr184

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4
Q

How does aequorin bind to calcium?

A

3 EF hand domains bind to calcium

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5
Q

Luminescent proteins

A

Dissipate chemical/mechanical energy by emitting the energy as a photon

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6
Q

Fluorescent proteins

A

Absorbs photon/light energy to become excited and then emits a 2nd photon to dissipate the energy (GFP)

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7
Q

Excitation

A

Wavelengths absorbed

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8
Q

Emission

A

emits light

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9
Q

Which always has lower energy excitation or emission?

A

Emitted photons because some energy is lost

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10
Q

Green Fluorescent Protein (GFP) structure?

A

-tight11 beta barrel
-chromophore is at center if barrel
-hydrophobic beta barrel protects chromophore from ions and excess oxygen

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11
Q

Wha3 residues is GFP’s chromophore made of?

A
  1. Ser65
  2. Tyr66
  3. Gly67
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12
Q

How do GFP mutations change color?

A

These mutations alter the energy
input required for excitation and the
energy output released upon emission

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13
Q

DsRed

A

-fluorescent red protein
-tetramer
-prone to slow folding and misfolding
-33 amino acid substitution needed to make mRFP1 (first red protein)

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14
Q

mCherry

A

-optimized version of mRFP1
-more intense color

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15
Q

How is a calcium ion sensor made?

A

It is made by fusing mutant GFP to calmodulin

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16
Q

How is the intensity of the fluorescence emission manipulated?

A

-when Ca2+ binds to calmodulin the conformational change makes gap in the GFP barrel structure so fluorescence is no longer quenched and GFP fluoresces

17
Q

What are fluorescent timer proteins and how do they work?

A

-track age of proteins in the cell
-when 1st expressed it is one color but then changes to another color due to chromophore oxidation

18
Q

What is FRET

A

-Fluorescence Resonance Energy Transfer, which uses 1 fluorescent protein to excite another
-only happens if proper wavelength is used and close in proximity

19
Q

What color is fluorescence when CFP is far from YFP?

A

Cyan fluorescence
-proteins do not interact

20
Q

What color is fluorescence when CFP is close to YFP?

A

Yellow fluorescence
-proteins do interact

Biochemistry (30 decks)

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