Re: [ccp4bb] off topic Thermal shift assay

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

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Subject: Re: Protein concentration vs Molecular wt...
From: "Oganesyan, Vaheh" OganesyanV {- at -} MEDIMMUNE {- dot -} COM
Date: 2012-07-20

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From: RHYS GRINTER r {- dot -} grinter {- dot -} 1 {- at -} RESEARCH {- dot -} GLA {- dot -} AC {- dot -} UK
Date: 2012-07-20

Subject: Re: off topic Thermal shift assay
From: "Dr {- dot -} Lorenzo Finci" lfinci {- at -} HOTMAIL {- dot -} COM
Date: 2012-07-20

Noor,
Thank you very much for your inquiry. As we all know, thermodynamic principles of cooperativity and allostery have long been used as a foundation to begin understanding the complex interplay between associated ligand binding events. In principle, the delta Tm shifts that occur when multiple ligands bind to the same protein should further manifest cooperative effects between the inherent binding sites. A unique property attributed to the Thermofluor is that it offers a high throughput approach to the study of allosteric interactions between protein and ligand. In terms of unfolding, Thermofluor has the capability to answer whether the flexibility of the protein is expressed as the number of different stable conformational states in high or low quantities. This is due to the fact that the range of temperature that unfolding occurs is reported by the flexibility of the protein. For example, steep transitions are indicative of highly cooperative unfolding, whereas shallow transitions indicate high flexibility. Multidomain proteins reflect an observed monophasic unfolding transition, and this is what is generally accepted as two-state unfolding. More complex unfolding transitions reflect that unfolding of the domains does not occur in a concerted manner. In order to obtain a detailed understanding of the linkage between ligand binding and protein stability, a concert of biophysical characterization utilizing Thermofluor, ITC, and DSC should be utilized...
Refferences:1. Binding Techniques to Study the Allosteric Energy Cycle; Allostery: Methods and Protocols, Methods in Molecular Biology, 2012, Kranz et al2. http://.thermofluor.org3. http://thermofluor.org/resources/Niesen-fingerprinting_Oxford.pdf4. Thermodynamic Stability of Carbonic ANhydrase: Measurements of Binding Affinity and Stoichiometry Using Thermofluor, Biochemistry, 2005, Matulis et al
Sincerely, lorenzo

Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China

Date: Thu, 19 Jul 2012 21:23:59 +0100
From: mohamed.noor@ul.ie
To: lfinci@HOTMAIL.COM
Subject: Re: [ccp4bb] off topic Thermal shift assay

Dear Lorenzo

> a measure of protein cooperatively,

Regarding
your comment on positive cooperativity, is there any literature
on this? I was taught that positive cooperativity for enzyme
will require a steady-state kinetic assay. How does this relate
to protein unfolding as measured by thermal shift?

Sorry for a basic question.

Thanks.

Regards

Mohamed

Mohamed Noor

Chemical and Environmental Sciences Department

University of Limerick

Ireland

On 19/07/2012 16:25, Dr. Lorenzo Finci wrote:

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

Previous message:
Subject: Re: Protein concentration vs Molecular wt...
From: "Oganesyan, Vaheh" OganesyanV {- at -} MEDIMMUNE {- dot -} COM
Date: 2012-07-20

Next message:
Subject: Ferredoxin Containing Crystal Bleaching
From: RHYS GRINTER r {- dot -} grinter {- dot -} 1 {- at -} RESEARCH {- dot -} GLA {- dot -} AC {- dot -} UK
Date: 2012-07-20