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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, [log in to unmask] 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



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Brett Collins, PhD
Group leader
Institute for Molecular Bioscience 
Level 3 North 
Queensland Bioscience Precinct
The University of Queensland
St. Lucia, 4072, Qld,
AUSTRALIA

phone: 61-7-3346-2043
FAX: 61-7-3346-2101

Courier address:

Institute for Molecular Bioscience
Queensland Bioscience Precinct
Building 80, Services Road
University of Queensland
St. Lucia, Brisbane
Queensland, Australia 4072
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