Hi Brett,
As Filip pointed out, pulldown assay is not an equilibrium
experiment. In addition, one of protein should be attached to the
resin in the pulldown assay, which might not be sometimes favorable.
You might not want to make very quantitative analysis on the pulldown
results.
If I were asked to choose one, I would believe ITC measurements. Can
you try other biophysical tools in order to cross-check binding
affinities?
Young-Tae
On Sep 2, 2008, at 3:13 PM, Brett Collins wrote:
Dear Artem,
Sorry I didn't want to scare people with too may details.
The pulldowns and ITC were done with the same protein samples
expressed in bacteria and purified by the usual ways. For both
mutant and wildtype the stoichiometry from ITC was approximately
1:1 (from about 0.85 to about 1.15 range). The ITC experiment was
performed over a period of an hour using 18 injections (i.e about
180 second delay between injections and the signal clearly reached
baseline again between injections). The pulldowns were perfomed
over a similar time period using several washes of His tagged
protein + ligand protein bound to beads.
Perhaps the only clear difference is that the pulldowns were done
in 20 mM Hepes pH 7.5 while the ITC was done in 20 mM Tris pH 8.0
(both with 150 mM NaCl, 1mM DTT). I imagine that the pH may make a
difference - but I am not convinced by this explanation. I would
still expect to see qualitatively the same result (reduction in
binding) at slightly higher pH.
Thanks,
brett
On 03/09/2008, at 7:00 AM, [EMAIL PROTECTED] wrote:
Dear Brett,
What was the value of N in your experiments? Did you get a clear "1"
(well, typically 0.8-0.9 is good enough)? Were there differences in N
between the two experiments? (Probably not but I'd rather ask).
What was peak integration time for the ITC experiment, and how
does it
compare with incubation time used in pull-downs?
How exactly were the pulldowns set up? Did you use pure protein or
was it
an extract of some sort?
Cheers!
Artem
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
################################
