What are the ways (in cell or cell free systems) through which we can experimentally prove that a chemical compound/drug molecule leads to complex formation between two proteins? I did Coimmunoprecipitation to prove this, but it did not work. I am afraid that the ternary complex (ProteinA + Compound + protein B) might have disrupted during cell lysis? To avoid this possibility, I am also doing alternate experiment where I incubated the chemical compound with cell lysates (instead of treating the cells followed by cell lysis) in order to allow for complex formation. Although the results of this experiment are awaiting, meantime could anyone suggest me something better?
I did not understand your question. I think your question needs to be more specific. Do not you think so? What is the mechanism by which the compound can mediate the interaction of the two proteins? Will it act as a linker? Will it act as an enzyme for complex formation? If you make the role of your compound more specific, then the scientific community can easily give you an idea for designing an experiment to test your thoughts using in vitro or in vivo models.
There are many ways to do this, especially if you have access to the purified proteins. However, for any binding experiment to work, the protein concentrations must be high enough that the equilibrium favors binding. In the case of the compound that mediates binding, the protein concentrations must be set so that binding will occur in the presence of compound but not in its absence.
Some techniques that can be used to test for protein-protein interactions:
- Co-immunoprecipitation (already tried unsuccessfully)
- Native gel electrophoresis
- Size exclusion chromatography
- Surface plasmon resonance
- Microscale thermophoresis
- Chemical crosslinking followed by SDS-PAGE
- Analytical ultracentrifugation
- Fluorescence resonance energy transfer
With purified proteins, I think I would start with the least sophisticated technique, native gel electrophoresis. Titrate the protein concentration, using a 1:1 ratio of the two proteins, with and without inhibitor, and look for a change in the migration rate of the proteins in the gel. The technique is simple and easy to do, does not require specialized equipment or training, does not require any labeling of the proteins, and uses very little protein. It can probably be done even with crude extracts if you use immunoblotting for detection.
I also like chemical crosslinking using BS3, a water-soluble crosslinker of primary amino groups. SDS-PAGE can be used to test for crosslinking of the two proteins. You only have to decide on a crosslinker concentration and then titrate the concentrations of the proteins, keeping them in a 1:1 ratio, with or without the compound.
Hi there,
If you have one of the proteins his tagged, you may immobilized it on NiNTA and then apply the other protein in presence or absence of the compound and see what can be eluted with imidazole. If protein coelution occurs only when chemical compound is present then interaction is dependent on the the chemical.
ELISA inspired approach may also be considered if you have a specific antibody for the partner in hand (coating the well with the first protein, applying the partner in presence or not of the molecule and finally detect the Partner with the specific antibody).
If your small molecule is cell-permeable (or endogenous to yeast), the yeast three hybrid system might be a good choice for you. We have used it to study small molecules that dimerize proteins. Here is a reference: Muddana, S. S; Peterson, B. R. “Facile Synthesis of CIDs: Biotinylated Estrone Oximes Efficiently Heterodimerize Estrogen Receptor and Streptavidin Proteins in Yeast Three Hybrid Systems” Org. Lett. 2004, 6, 1409-1412.
Another way to analyse protein association is low angle light scattering (LALS). This and SPR would be my first choices, because testing a large number of conditions (required for kinetics) can be done quickly.
It is really dependent on the interaction properties of your complex such as the bond strength, membrane/soluble proteins, interaction interface location et al.
It will be easy if you have both pure proteins. You can use SEC, SPR, OCTET et al. It will give you direct proofs whether two proteins interact with each other with the small molecule. If you don't have pure proteins and the interaction is strong enough, you can introduce one protein with tag and IP down to check by western blot or you can use one target antibody to IP down. The challenges for both method are that you may interfere the binding by introducing tag or antibody in the complex. If the interaction is very weak between two protein, SILAC/FRET may be a good choice, because it doesn't need to wash 3XPBS and most weak bounds will be washed away with 3XPBS.
I am afraid to tell that in my opinion, based in my experience, a synthetic "small" compound will not be a key factor to make two proteins to stick together. I apologize if I did not interpret well the question.
Actually, the PROTAC approach attempts to do exactly that, by using small bifunctional molecules to link a target protein to a ubiquitin E3 ligase (https://www.ncbi.nlm.nih.gov/pubmed/22350917).
Hi Adam
I do work with bivalent compounds that cannot make 2 proteins stick together. They may bind and de-bind but not allow formation a stable ternary complex. Exceptions of course ma occur.
Protein-protein interaction is always a challenge project. Depending on your materials (purified proteins or cell based assay) and requirement(if you need to know Kd or just want know if they interact or not), there are a list of methods. For quantitative results with purified proteins, fluorescence is the best method for its sensitivity and easy operation. If affinity is low, NMR is preferred. I don't recommend Surface plasma unless you can do site specific labeling to you protein. you may check our website for detail discussions
- Badges
- Science method