Protein interactions- none, now what???

John Kuszewski johnk at
Fri Apr 7 11:33:08 EST 1995

In article <3m1qdh$5eu at>, djdlab at (Dean Danner Lab) writes:
|> So- I would like to rack everyone's brain. 
|> I have proteins interacting with themselves (ie X with X, and Y with Y) in the yeast two hybrid system, and functioning in vivo in a yeast model system.
|> SO what if I make a mutation (deletion or single aa change) and the protein no longer interact of function?  What would be the best way to assess why I have lost interaction/function.  The proteins I am studying function is a multienzyme complex so they f|> orm 24mers, or tetramers depending on which ones we are talking about.

(Please try to limit yourself to 80 characters/line!)

If you want to know if your mutation has destabilized each monomer so 
much that it's unfolded the protein, have a look at the CD or NMR
spectra.  Calorimetry would also show changes in the stability.

If you want to see if you've just destabilized the interaction surfaces,
do some ultracentrifugation or light scattering measurements of the 
monomer-polymer equilibria.

If you see no changes in the free energy of monomer-monomer interactions
and you haven't unfolded the protein, then perhaps your mutations
are right in the active site.

Hope this helps.

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