Hi Brett,

Your kinetics is most likely different now.  Pull downs are affected by kinetics: fast kinetics interactions are harder to detect by pull-downs, and such interactions may look weaker even though the affinity is identical. The fact that changed some of your interface and ended up changing enthalpy/entropy, there is a good chance that kinetics is changed as well.

For a more intuitive understanding, think about this: When you are washing your pull-downs, you are more likely to lose faster k-off interactions, than the slower.  During washing, there is almost no associating, but only dissociating, which is a function of k-off, not Kd). ITC is an equilibrium experiment and the number you get there is believable, barring any changes in buffer conditions, temperature, etc.

You might want to measure kinetics with SPR to put this one to rest. That would also be a full characterization of your mutant (i.e. kinetic and thermodynamic).

Engin

----- Original Message -----

From: "Brett Collins" <[log in to unmask]>

To: [log in to unmask]

Sent: Tuesday, September 2, 2008 1:37:41 PM GMT -08:00 US/Canada Pacific

Subject: [ccp4bb] Puzzling protein-protein interaction

Dear CCP4 Community,

My apologies for the non-crystallography biochemical 

question but it occurred to me that there are many people 

on this list who are also very good biochemists.

We have just performed an ITC experiment with two proteins 

and measured a Kd of 150 nM, deltaH of -15 kCal/mol, 

deltaS of -15 Cal/mol/K and deltaCp of -2000 J/Mol/K.

We also measured the binding of a mutant of one of these 

proteins predicted from crystal structure to inhibit 

binding of a small fragment of peptide (this is predicted 

to reduce binding slightly but not to affect total binding 

as there is still a large interaction interface that is 

left intact).

This mutant has a Kd of 150 nM as well, but deltaH is -10 

kCal/mol, delta S is essentially zero, and deltaCp reduces 

in magnitude to about -1500 J/Mol/K as we would predict 

from the change of buried surface area. The ITC data looks 

good and we have repeated the experiments a number of 

times so they are statistically significant. The 

experiments were performed within reasonable concentration 

limits (~10uM protein in the cell so the C-value is about 

50-100)

Now the puzzle is that the mutant binds less strongly in 

pulldowns (about 50% reduction after several washes) but 

we see an almost identical Kd by ITC despite major changes 

in enthalpy/entropy contributions to binding. The mutant 

and wildtype appear to have identical fold by CD but of 

course there may be small differences. Everything makes 

sense except the lack of Kd change by ITC.

Does anyone have any experience of similar results, or 

more importantly have a possible explanation for them?

Any thoughts greatly appreciated.

Brett Collins