Question

While perusing the scientific literature late one night, you come across a report that a protein called Thingamajig is thought to be involved in the development of antibiotic resistance, an emerging global health crisis. This putative involvement appears to involve an interaction with another protein, Whosiewhatsit. You would like to study Thingamajig, and its interaction with Whosiewhatsit, but neither protein has been cloned.

a) How would you set out to study these proteins and their interaction? Describe in general terms (a flowchart, for example), filling in the details once this framework is assembled. To get you started, how would you obtain the information necessary to express and purify these proteins, and how would you manipulate this information?

b) Assuming Thingamajig is positively charged and Whosiewhatsit is negatively charged, what purification approach might you use to purify each of them independently? What approach might you take to purify the complex of the two?

c) What approach would you take to identify the amino acid residues — in both Thingamajig and Whosiewhatsit — that underlie their interaction with one another?

Answer 1

A) The best ways to detect these two proteins interaction are:

1. Coimmunoprecipitation: It can confirm interactions using a whole cell extract where proteins are present in their native form in a complex mixture of cellular components.
2. Tandem affinity purification-mass spectroscopy (TAP-MS): TAP-MS is based on the double tagging of the protein of interest on its chromosomal locus, followed by a two-step purification process and mass spectroscopic analysis.
3. Affinity chromatography: Affinity chromatography is highly responsive, can even detect weakest interactions in proteins.

To express and to purify these proteins we must have to obtain:

1. The gene sequence of the proteins from where they are transcribed.
2. The types of the proteins whether they are membrane-bound or globular.
3. The stability of the proteins means in which buffer and in which temperature these proteins doesn't precipitate.
4. The expression of proteins, whether they are secretory or they are inclusion bodies.

B) As 'Thingamajig' is positively charged then the purification technique to purify Thingamajig must be 'Ion Exchange Chromatography' and the beads which will use to purify the protein must be positively charged beads. Example: Q column and DEAE beads.

As Whosiewhatsit is negatively charged, then the purification technique to purify Whosiewhatsit must be 'Ion Exchange Chromatography' and the beads which will use to purify the protein must be negatively charged beads. Example: S column.

If we have to purify the complex of the two proteins then Size exclusion chromatography, Gel filtration Chromatography or Fast Protein Liquid Chromatography (FPLC) is the best way to purify the protein complex.

C) Edman's Degradation is the possible way to detect the amino acid residues of those proteins which interact with each another. But the best way to detect the interacting portions of these proteins is to use some Computational tool (Example: GROMACS) to detect the protein-protein interacting amino acids residue.