[ccp4bb] Puzzling protein-protein interaction

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: about pattersom map and self rotation function
From: Ron hudson hudson {- dot -} genome {- at -} GMAIL {- dot -} COM
Date: 2008-09-03

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From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2008-09-03

Subject: Puzzling protein-protein interaction
From: Brett Collins b {- dot -} collins {- at -} IMB {- dot -} UQ {- dot -} EDU {- dot -} AU
Date: 2008-09-03

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

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: about pattersom map and self rotation function
From: Ron hudson hudson {- dot -} genome {- at -} GMAIL {- dot -} COM
Date: 2008-09-03

Next message:
Subject: PX beamtime application deadline Sept 3rd!
From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2008-09-03