Dear all,
Many thanks for all the responses to my question. It seems the
consensus is that the differences we are seeing between ITC and
pulldowns are most likely due to a change in the kinetics of the
interaction for the mutant protein. i.e with pulldowns if the Koff is
now faster we will see an apparently weaker binding as protein is
lost. See the below comments by Engin which I think summarise the
main thrust of the comments I've had very nicely.
Thanks again,
Brett
On 03/09/2008, at 3:24 PM, Engin Ozkan wrote:
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" <[EMAIL PROTECTED]>
To: [email protected]
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
################################
Brett Collins, PhD
Group leader
Institute for Molecular Bioscience
Level 3 North
Queensland Bioscience Precinct
The University of Queensland
St. Lucia, 4072, Qld,
AUSTRALIA
e-mail: [EMAIL PROTECTED]
phone: 61-7-3346-2043
FAX: 61-7-3346-2101
website: http://www.imb.uq.edu.au/index.html?page=82433
Courier address:
Institute for Molecular Bioscience
Queensland Bioscience Precinct
Building 80, Services Road
University of Queensland
St. Lucia, Brisbane
Queensland, Australia 4072
################################
