Re: [ccp4bb] off topic Thermal shift assay

[Artem Evdokimov]

Just a quick note: there is a thermal shift-like methd that is still htp
and allows one to measure unfolding of membrane proteins etc. The method
relies on a cys-reactive leuco dye that becomes fluorescent (or colored)
after it reacts with an sh. As long as your protein has at least one buried
SH this will work more or less regardless of conditions (might be less
effective at low ph, don't add dtt or bme, yadda yadda

I used it and it is very nice. Hope this helps.

Arem
On Jul 20, 2012 11:03 AM, "Dr. Lorenzo Finci"  wrote:

>  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 al
> 2. http://.thermofluor.org
> 3. http://thermofluor.org/resources/Niesen-fingerprinting_Oxford.pdf
> 4. 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 Laboratory
> Harvard Medical School
> Dana-Farber Cancer Institute
> Boston, MA
> Peking University
> The College of Life Sciences
> Beijing, 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:
>
>

Re: [ccp4bb] Nucleophilic attack by the side-chain carboxyl group of Asp?

[Justin Jones]

Unfortunately, their isn't a "general rule" for good versus bad
nucleophiles. However, their are some key classical examples, which are
reviewed extensively in enzymology textbooks. Remember, that a nucleophile
is dependent on it's pKa value, which depends on the surrounding residues.
Thus, two different enzymes that possess the same type of nucleophile but
perform different reactions can have different pKa values, and therefore
different rates. It is also dependent on the leaving group potential of
the electrophile and the stability of the covalent intermediate. Some
examples of nucleophiles in no particular order include, serine, cysteine,
aspartate, lysine, histidine, and tyrosine. Hope this help.

Justin

 

On 7/21/12 1:03 AM, "Peter Hsu"  wrote:

>I too am also studying the reaction mechanism of an enzyme, but my
>chemistry/enzymatic biochemistry is rather weak after many years of
>non-use and no review. Does anyone know just as a general rule which
>residues are the best to worst nucleophiles?
>
>Sorry if this seems rather presumptuous, just not sure where to look in
>literature for a summary of these things.

Re: [ccp4bb] Nucleophilic attack by the side-chain carboxyl group of Asp?

[Roger Rowlett]

Biochemistry and enzymology texts are a good place to start. In general,
stronger bases are better nucleophiles (it doesn't correspond exactly) but
you also have to consider the population of the deprotonated species at the
relevant pH, and the role of neighboring residues in altering 'native' pKa
values of ionizable residues and the local dielectric constant.

Is your Asp hypothetically acting as a nucleophile, or as a general base?
The latter is a more common role for Asp. His, Cys, or metal-hydroxide are
more common nucleophilic catalysts: these residues are both good
nucleophiles and can act as decent leaving groups.

Roger Rowlett
On Jul 21, 2012 1:05 AM, "Peter Hsu"  wrote:

> I too am also studying the reaction mechanism of an enzyme, but my
> chemistry/enzymatic biochemistry is rather weak after many years of non-use
> and no review. Does anyone know just as a general rule which residues are
> the best to worst nucleophiles?
>
> Sorry if this seems rather presumptuous, just not sure where to look in
> literature for a summary of these things.
>