Protein melting temperatures

From: Patrick Shaw Stewart
Date: 28 September 2011 12:25

I actually think you can make comparisons between different proteins.  We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently.  

Basically, 45C seemed to be the dividing line.  If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures.

Patrick

On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis wrote:

Hello -

The excellent paper of McCrary, uses differential scanning 
calorimetry, which will give an absolute measure of thermostability.

Using Thermofluor I would be afraid you can only assess the relative 
thermostability of one protein in different conditions.
As your fluorescence reporter would interact differently with exposed 
hydro[hobic patches in different proteins, I would be a bit more careful
in comparing the Thermofluor results between different proteins ... I 
am not aware of anyone correlating differential scanning calorimetrywith
Thermofluor data, but I must admit I have not looked up that 
literature recently.

A.


On 23 Sep 2010, at 18:40, Philippe DUMAS wrote:

> Le 23/09/2010 17:28, Raji Edayathumangalam a écrit :
>
> Raji
> I suggest having a look to this paper:
> McCrary et al. J. Mol. Biol. 264(1996) 784
> where you will find an interesting study on protein stability and an
> interesting comparison with other proteins.
> Philippe Dumas
>
>> Hi Folks,
>>
>> Sorry for the pre-xtallo question; pre-xtallo right now, but hoping 
>> to
>> take my protein the xtallo way one of these days!
>>
>> I am currently performing Thermofluor assays with my protein and the
>> results show that the Tm is ~45C.  I am looking for some examples of
>> proteins and their melting temperatures so that I can gauge where my
>> protein falls in the spectrum of unstable-to-stably folded. For
>> example, the melting temperature of some forms of lysozyme is 73.8C
>> (very stable, I suppose).
>>
>> Just need a sense for whether my protein is considered unstable or
>> somewhat stable. Please could you share some examples.
>>
>> Many thanks.
>> Raji
>>
>> -----------
>> Raji Edayathumangalam
>> Joint Research Fellow
>> Harvard Medical School/
>> Brigham and Women's Hospital
>> Brandeis University
>>
>
> <McCrary-JMB264(1996)784.pdf><p_dumas.vcf>

--

----------
From: Artem Evdokimov

For what it's worth, we've been using thermofluor to compare the 'apparent' melting points of enzymes with their thermal stability measured as inhibition of their respective reactions by elevated temperature. The data so far make sense - the differences in apparent enzyme Tm (using the same conditions as the reaction mix!) match the differences in the half-inhibition T. Not the absolute number,though (which is not unexpected givn the different kinds of measurements involved).

 

So I'd say thermofluor is reasonably good at comparing different proteins. Qualitatively at least.

 

Artem

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From: Bosch, Juergen

We have proteins that melt at >60˚C but they don't crystallize. According to your 45 degree rule we should have crystals, what are we doing wrong ?

Jürgen

......................

Jürgen Bosch

Johns Hopkins University

Bloomberg School of Public Health

Department of Biochemistry & Molecular Biology

Johns Hopkins Malaria Research Institute

615 North Wolfe Street, W8708

Baltimore, MD 21205

----------
From: Linda Schuldt


Dear Raji,

what exactly do you mean when you say the melting temperature is 45deg.
Did you only test one buffer, or did you test many buffers and 45deg is
the most stable one? If you have only tested one buffer you should run a
screen testing different buffer systems (pH) and e.g. NaCl concentration
and glycerol concentrations (or ligands, if your proteins binds any). Then
you identify the buffer which is stabilizing your protein the most. I have
seen big impacts on protein stability and crystallization when optimizing
my buffers like this.

I think you should not only consider the melting temperature alone, but
also how the curve looks like. Do you get a high initial flourescence
(which often indicates partially unfolded protein or hydrophobic patches)
or do you have very low initial flourescence (which is a good sign for
compact protein). Another thing to look at is if your transition is sharp
(the steeper the better). Taking all this together you can judge if your
protein is happy or not.

Hope this helps you!

Linda

Patrick Shaw Stewart wrote:
> I actually think you *can *make comparisons between different proteins.
>> **

*******************************
Dr. Linda Schuldt
Department of Molecular Biology
University of Aarhus
Science Park
Gustav Wieds Vej 10c
DK-8000 Århus C
Denmark

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From: sankaranarayanan srinivasan

This paper on thermofluor is a good reference and if you have access to a real time PCR machine, different buffer systems, like the PACT screen can be evaluated within an hour to find out the buffer in which your protein is most stable.

It gives the Tm of your protein and if you have a high fluorescence to start with, it means your protein is unfolded to start with.

Curr Protoc Mol Biol. 2011 Apr;Chapter 10:Unit10.28.

The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization.

Phillips K, de la Peña AH.

Best regards

Shankar

----------
From: Patrick Shaw Stewart


Susan and everyone,

I should apologise for any confusion that I may have caused.

Rajiv actually asked his question a year ago, and I accidentally replied to it a year too late!

It's an interesting question though

Patrick

> I actually think you *can *make comparisons between different proteins. >> **