[ccp4bb] SUMMARY - 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

Previous message:
Subject: Re: Building peptide in density using coot.
From: Chavas Leo ccp4hnaalas {- at -} GMAIL {- dot -} COM
Date: 2008-09-03

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Subject: Re: Building peptide in density using coot.
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2008-09-03

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

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"
> To: CCP4BB@JISCMAIL.AC.UK
> 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: b.collins@imb.uq.edu.au
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
################################

CCP4bb navigation

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

Previous message:
Subject: Re: Building peptide in density using coot.
From: Chavas Leo ccp4hnaalas {- at -} GMAIL {- dot -} COM
Date: 2008-09-03

Next message:
Subject: Re: Building peptide in density using coot.
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2008-09-03