Protein-Protein interaction techniques

Question 1

Yeast two hybrids

Answer 1

Depend on Gal 4 transcription factors’s DNA binding domain and activation domain that binds to RNA polymerase to initiate transcription.

Bind protein X to Gal4 (bind domain)
Express the activation domain fused to protein Y

X binds to Y, DNA binding domain is brought together with the activation domain, resulting in expression of reporter gene.

This is used for yeast that cannot make histidine.
Tyrp and Leu is also excluded for a control.

Question 2

Cell degredation - S7

Answer 2

S7 binds to mbm2. mbm2 codes for the breakdown for p53 cancer controlling protein. S7 inhibits this process.

Question 3

Immunofluorescence

Answer 3

Finding out where proteins are in the cell.

Marking protein of interest (e.g nucleus, ER)

Process:
Glass slide sample
Fix sample, cold methanol.
Permeabilise membrane to acess organelles.

Add antibody > Wash
Add secondary antibody
2 species are displayed fluorescently

Question 4

Green fluorescent protein (GFP)

Answer 4

B-barrel structure, from jellyfish

Fuse GFP to protein of interest (transfecting cell)
by using gene, GFP fusing protein .

Varions have been made, blue,green,cyan,yellow. each have a different optimal fluor wavelength,

Question 5

Fluoresence resonance energy transfer (FRET)

Answer 5

Emission of first protein overlaps with the excitation of the second protein, they must have overlapping fluorescent wavelenghths.

Fluors need to be within close proximity!!

Process: Protein A will absorb a wavelength (precise), the emission spec is absorbed by the second protein (B) the fluoresence is emitted. The fluor given out is the same from the first protein.

Question 6

Biacore

Answer 6

Protein is immobilised on a chip and a solution containing a different protein is passed over it

Result:
If the protein binds to the immobilised protein, a parameter of the chip called surface plasmon resonance changes

This quantifies affinity of the interaction.

The glass slide contains a gold layer, containing a bait protein, this is bathes into a solution with protein of interest.

Light is shone into the gold layer at an angle, this excites the electron to move up and down. This angle is specific (resonance angle)

If protein of interest binds to bait protein, the electron flow in gold layer is interrupted and the resonance angle is changed.

Question 7

Biacore trace

Answer 7

Sortillin, a protein which inhibits binding of receptors by covering the binding site by using its own precursor protein containing propeptide

Experiment:
Mature sortilling in biacore as bait protein
Bathed in propeptide (target protein)
GST does not bind, hence precursor form of sortilin is autoinhibited.