Question

3. You have just done a proteomics experiment investigating protein-protein interactions and you have a couple good leads.

A) How might you use microscopy to verify potential interactions between proteins? Name the type of experiment and explain the principle upon which it is based. (3 marks)

B) Propose how dyes will be incorporated into your sample, specify which dyes you would use, defining the excitation and emission wavelength of the dyes. (3 marks)

C) Explain the expected results if there is, or is not, an interaction. (2 marks)

Answer 1

Ans A) Fluorescence microscopy technique can be used to study protein interactions. When the molecules are illuminated by the UV light,the molecules absorb energy and go to excited state and emit visible radiations while coming back to the ground state while releasing the energy.This phenomenon is known as Fluorescence.The emitted light is always of longer wavelength than the absorbed light (Absorbed light has high energy or Less wavelength,when the molecules come to ground state while releasing energy,the emitted light has lesser energy or Longer wavelength).

Ans B) Commonly used fluorescent dyes are Fluorescein (which emit green fluorescence when excited with blue light) and rhodamine (emits red fluorescence when excited with green-yellow light). The sample is treated with the wavelength of light corresponding to the excitation wavelength of respective fluorescent dyes with the help of excitation filter which permits only the wavelength of light that excites particular fluorescent dye.The molecules absorb this wavelength,gets excited and released fluorescence or emission wavelength which can be recorded as a fluorescence intensity with the help of detector.

Ans C) Lets us understand the protein interaction experimentally.

In cell signalling of G-Proteins, G-alpha subunit is bound to CFP dye (Emission of CFP is at 490nm) and G-beta-gamma subunit is bound to YFP or yellow fluorescent dye (Emission of YFP is at 527nm). If there would be interaction between G-alpha and G-beta-gamma subunits,FRET (fluorescence resonance energy transfer) will occur). After excitation wavelength of 440-490nm is passed (440-490 is excitation wavelength of CFP dye),emission will occur at 527nm approx. which is the emission wavelength of YFP dye which means that CFP gives energy to the YFP dye,then the YFP dye absorbs energy and emits at 527nm which means that FRET occurred which in turn means that interactions of proteins have taken place.

We know that after ligand binding, G-beta-gamma dissociates from G-alpha subunit.So now when excitation wavelength (440nm) was passed,there was emission of cyan light of CFP and not yellow light of YFP because these is no interactions between proteins and thus no FRET occurs (since CFP cannot give energy to YFP due to lack of interactions between the subunits of G-protein).