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

Protein - protein interaction (PPI) studies include vivid methods. As which methods have been used is not mentioned, assuming any method that has been used to check PPI gave significant positive result about their interaction, I'll answer following questions.

(A) To verify PPI using microscopic method:

Proteins can be small or large in size, hence, the type of microscopy to determine the protein depends on its size. Also, if proteins are to be analysed in living condition Atomic Force Microscopy (AFM) can be used. Other method that you can use is electron microscopy to analyse protein's structure (transmission cryoEM).

To specifically determine PPI interaction using microscopy,

(i) using AFM- suppose their are two proteins A and B that are suspected to bind to one another to perform a function. To check whether these proteins interact or not, first you need to check the structure of protein A and B in isolation using electron microscopy. After that you can mix both the protein samples to check if they react to one another. If they react their structures might change and the binding might alter the distance between the two proteins initially and after binding which can be measured.

(ii) AFM-FRET - In FRET their is a donor molecule which imparts its fluroscence to the acceptor molecule; based on the information of fluoroscence transfer, proximity of the two molecules can be known. A high end technique such as AFM-FRET has been reported in s study where the AFM tip contains a molecule that has acceptor dye and the sample has molecule of donor dye. Donor dye will donate its fluorescence from the sample to the acceptor dye which is present on the tip and the resultant changed emission spectra from the tip can be detected. This emission spectrum is strongly dependant on distance. So, if the molecules interact and thus are in proximity, they'll have a different emission spectrum and of they dont interact, they'll result into different spectrum.

(B) In an in vitro study, the gene expressing protein A can be recombined to a reporter gene such as YFP and the the gene expressing protein A can be recombined to a reporter gene such as RFP. Excitatation range of YFP is 490-510nm and emission range is 520-550nm. Simillarly, excitatation range of RFP is 558nm and emission range is 583nm. In a technique such as FRET, if two proteins expressed by these reporter genes, and if they interact, then resultant fluoroscence will be orange, if not the resulatant fluorescence will be red (assuming RFP is acceptor and YFP is donor).

(C) If protein A and B interact, then as metioned in (A), that the signals for the emission will alter in case of microscopy and it can be detected. Further, it can be cross checked using techniques such as yeast II hybrid systems, co-immunoprecipitation, etc.