Lecture 27 - GFP Flashcards
What is the visible light spectrum?
- the segment of electromagnetic spectrum that the human eye can view
- shorter wavelength = more energy (i.e., longer wavelength = less energy)
What is fluorescence?
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
Fluorescent molecules
- absorb light at one wavelength and emit it at another, longer wavelength
How does a fluorochrome get excited? How does fluorescence work?
- 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
What happens if there is too much light?
too much light exposure destroys the fluorochrome molecule in a process called photobleaching
- molecule can no longer be seen
What is immunofluorescence?
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)
Where does the original green fluorescent protein come from?
- 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
How do aequorin and GFP work together?
- to convert Ca2+- induced luminescent signals into the green luminescence
Why can GFP be entirely genetically coded? What is the chromophore made of?
because it does not need a prosthetic group or any other cofactor
- chromophore is made by amino acids (chromophore = part that is excited)
What does fusing GFP to the coding sequence of a gene allow?
direct visualization of the protein
- fuse the GFP gene to another gene and both genes will be transcribed and translated together
What needs to be added to the GFP to direct it to a particular cell compartment?
a peptide location signal
- NLS-GFP
- Mitochondria-GFP
- KDEL-GFP
etc
What do mutations in GFP do?
- 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.
Can we track movement of proteins in vivo using GFP?
yes
Calcium and fluorescence
- 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)
GFP fluorescence responds rapidly and reversibly to ___ ______
pH changes
What is photobleaching? FRAP?
- 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)
What molecule extends the linker in the sensor molecule between 2 proteins? (FRET microscopy). Explain what happens on this slide
cAMP; cAMP extends the linker, making the proteins too far apart for energy transfer
FRET
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
Photoactivatable fluorescent proteins
- fluorescent proteins that display unique changes in their spectral properties upon exposure to a specific wavelength of light
How can we test the ‘age’ of proteins and cells?
- some fluorescent proteins slowly change their colour over time
FRET can be used to make genetically encoded ________ ________
fluorescent biosensors
Split GFP
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)
