I am working on a project to evaluate the interaction between two proteins, let's say X and Y. Y was found as a candidate protein to interact with X in a Y2H screen, where C-terminal pieces of X were used as baits. When I tried to verify the interaction with Pull-down or CoIP, I got the interaction of Y with both N- and C-terminal pieces of X, but never with the full-lenght X.
Now I wonder, what if the two full-length functional proteins do not actually interact in the cellular context! Is it normal that we may not see the interaction of the full-length proteins in vitro? I am also thinking if I should go forward with this candidate protein (functional assays in neurons). Please help me with your knowedge and expertise.
Protein interactions occur through particular domains, and these regions might still form meaningful interactions even if the full-length proteins do not. Proteins may undergo conformational changes that enable interactions in specific conditions or cellular compartments, revealing important regulatory mechanisms. Such studies can uncover alternative or transient interactions that are crucial for understanding cellular processes, signal transduction, and complex formation. This approach is also valuable in drug discovery, as targeting protein fragments or specific domains can lead to therapeutic interventions.
Do you know the structure of X? Does it make sense, structurally, that Y interacts with both N and C termini (i.e. can you imagine a conformational change that would lead to termini being exposed and next to each other?)? What else do you pulldown with Y, maybe it's just sticky? Are all those experiments done on lysates or have you ever expressed recombinant X? Just some things to think about.
Dear Sujan Bhowmick
are both soluble protiens or at least one of the 2 is a membrane protein? since the presence of the membrane region may affect your results
best
Manuelòe
did you look at the 3D structure of your X prot (alpha fold !) maybe you have two domains of X that interact with Y.
can you imagine a biological role for these interactions? if yes then it is worth to try to understand what happen .. your challenge will be to find which triggers X "opening" (phosphorylation?)
is Y a sticky protein? try interaction of Y with a Z protein; there are lists of false positives in 2HY.
do the coIP and pull down in the biological relevent context of your model if you work in mammalian cells for example then do it in mammalian cells.
You didn't mention proteins, but what if one of the two is a membrane protein? The tails of membrane proteins often extend out from the membrane and interact with other proteins. Alternatively, it could be a peripheral membrane protein characterized by a more rigid core and highly mobile, charged tails that serve as the interacting components. In this case, it would also be quite soluble in the cytoplasm.
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