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.n...@ul.ie
To: lfi...@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:
