Lecture 27 - GFP

Question 1

What is the visible light spectrum?

Answer 1

• the segment of electromagnetic spectrum that the human eye can view
• shorter wavelength = more energy (i.e., longer wavelength = less energy)

Question 2

What is fluorescence?

Answer 2

the emission of light by a substance that has absorbed light or other radiation
- one wavelength could excite a molecule, while another wavelength won’t

Question 3

Fluorescent molecules

Answer 3

• absorb light at one wavelength and emit it at another, longer wavelength

Question 4

How does a fluorochrome get excited? How does fluorescence work?

Answer 4

• an orbital electron of a fluorochrome molecule can be raised to an excited state after the absorption of a photon
• fluorescence occurs when an electron returns to its ground state and emits a photon of light at a longer wavelength

Question 5

What happens if there is too much light?

Answer 5

too much light exposure destroys the fluorochrome molecule in a process called photobleaching
- molecule can no longer be seen

Question 6

What is immunofluorescence?

Answer 6

a method commonly used in molecular and cell biology labs as a robust and simple method to reliably localize (i.e., find out where they are) molecules on fixed cells or tissues (i.e., dead)

Question 7

Where does the original green fluorescent protein come from?

Answer 7

• a jellyfish
• aequorin is a blue-light emitting bioluminescent protein
  -GFP is a green-light emitting protein
• GFP gets excited by blue light from auquorin => emits green light

Question 8

How do aequorin and GFP work together?

Answer 8

• to convert Ca2+- induced luminescent signals into the green luminescence

Question 9

Why can GFP be entirely genetically coded? What is the chromophore made of?

Answer 9

because it does not need a prosthetic group or any other cofactor
- chromophore is made by amino acids (chromophore = part that is excited)

Question 10

What does fusing GFP to the coding sequence of a gene allow?

Answer 10

direct visualization of the protein
- fuse the GFP gene to another gene and both genes will be transcribed and translated together

Question 11

What needs to be added to the GFP to direct it to a particular cell compartment?

Answer 11

a peptide location signal
- NLS-GFP
- Mitochondria-GFP
- KDEL-GFP
etc

Question 12

What do mutations in GFP do?

Answer 12

• they can change the absorption and emission colours
• can also change other fluorescence properties, like brightness stability, maturation times (i.e., how long it takes to fluoresce), etc.

Question 13

Can we track movement of proteins in vivo using GFP?

Answer 13

yes

Question 14

Calcium and fluorescence

Answer 14

• can use a fluorescent indicator to visualize intracellular calcium concentrations => GCaMP (GFP Calmoldulin Protein), measures Ca concentration
• calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo (changes in calcium = neuron activity)

Question 15

GFP fluorescence responds rapidly and reversibly to ___ ______

Answer 15

pH changes

Question 16

What is photobleaching? FRAP?

Answer 16

• the photochemical alteration of a fluorophore => makes it unable to fluoresce
• FRAP = fluorescence recovery after photobleaching
• indicates dynamics of a protein in a living cell (i.e., how fast proteins move)

Question 17

What molecule extends the linker in the sensor molecule between 2 proteins? (FRET microscopy). Explain what happens on this slide

Answer 17

cAMP; cAMP extends the linker, making the proteins too far apart for energy transfer

Question 17

FRET

Answer 17

fluorescence resonance energy transfer => a special technique to gauge the distance between 2 chromophores
- test how close 2 proteins are in a sample
- test protein to protein binding
- the emission wavelength of one protein is the excitation wavelength of the other protein => we detect the emission of the second protein

Question 17

Photoactivatable fluorescent proteins

Answer 17

• fluorescent proteins that display unique changes in their spectral properties upon exposure to a specific wavelength of light

Question 17

How can we test the ‘age’ of proteins and cells?

Answer 17

• some fluorescent proteins slowly change their colour over time

Question 18

FRET can be used to make genetically encoded ________ ________

Answer 18

fluorescent biosensors

Question 19

Split GFP

Answer 19

GFP proteins can be split into fragments that are attached to 2 separate proteins => spontaneously assemble into a functional protein when in very close proximity
- tells us about protein-protein interactions and cell-cell contacts (i.e., can know if presynaptic and postsynaptic neurons are in contact)