Dear Anita
One alternative method to determine the thermodynamics and potentially discern the folding energy changes from the interaction driven ones would be NMR. Advantages over ITC experiments include: determine if the interaction drives foldness, estimate associated thermodynamics and, if you do 3D labelling experiments and do backbone assignment, you can map the interaction interface from the NMR data. Elegant NMR experiments have been extensively exploited to study unfolded/folded states and interactions in recent times, with exciting results which proved to be unachievable by any other biochemical/biophysical/structural technique, so it was definitely worth trying!
Good luck
Paula
===================================
Dr Paula S. Salgado
Lecturer in Macromolecular Crystallography
Institute for Cell and Molecular Biosciences
Faculty of Medical Sciences
3rd Floor Cookson Building
Newcastle University
Newcastle upon Tyne, NE2 4HH, UK
Tel: +44 (0)191 222 7369
Fax: +44 (0)191 222 7424
Email: [log in to unmask]
________________________________________
From: CCP4 bulletin board [[log in to unmask]] on behalf of Anita P [[log in to unmask]]
Sent: 14 March 2014 13:57
To: [log in to unmask]
Subject: Re: [ccp4bb] ITC with unfolded proteins
That is a very interesting question, which I would request the seniors out there to give their insights on.
I was imagining that a recombinant purification of an unfolded partner would aggregate which would cause trouble in ITC. Am I correct in this theory?
Would love to have more insights.
thanks in advance
Anita
On Fri, Mar 14, 2014 at 7:18 PM, <[log in to unmask]<mailto:[log in to unmask]>> wrote:
Hi,
I think the experiment is doable, but how would you decouple
protein-protein interaction from folding of the unfolded
protein due to protein interaction?
Reza
Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY 10031
Tel. (212) 650-6070<tel:%28212%29%20650-6070>
---- Original message ----
>Date: Fri, 14 Mar 2014 18:07:48 +0530
>From: CCP4 bulletin board <[log in to unmask]<mailto:[log in to unmask]>> (on behalf
of Anita P <[log in to unmask]<mailto:[log in to unmask]>>)
>Subject: [ccp4bb] ITC with unfolded proteins
>To: [log in to unmask]<mailto:[log in to unmask]>
>
> Hello everyone,
> I have a query for the scientists working on
> protein-protein interaction.
> It is known that some proteins exist in unfolded or
> molten globule state and attain structure on
> interaction with other folded proteins.
> Many a times, it is difficult to obtain the
> structure of these complexes.
> Is it possible to quantitatively determine the
> thermodynamics of interaction between an unfolded
> protein and a folded protein using ITC? Later may be
> perform an alascan to determine the residues of the
> unfolded partner involved in the interaction.
> Please share your ideas
> cheers**
> Anita
|