Up on my knowledge, for determining thermostability of an enzyme there is no need to do DSC and DSC can not give the enzyme activity with respect to a particular temperature. If you are determining the pyrolysis pattern of a peptide you can use DSC. To do the thermostability you should estimate the activity at different temperatures. The specific temperature at which the activity diminish is the maximum temperature (not Optimum) and you should select different temperatures according to that. If you need more information you can contact in e-mail.

for DSC ask Dominique Madern, he knows verything about hyperthermophilic organims. We used CD spectra vs. temperature (not published)

Hi,

I was working with nano DSC, and through this technique I have been able to identify two different thermodynamic protein populations inside my protein sample. The only difference was that the protein was presenting an active and an inactive conformation. Through DSC we have determined the stability change and we have also investigated the role of the substrate in protein activation mechanism.

Alternatively you can do Circular Dichroism measurement at a fixed wavelenght varying the temperature to determined when conformational changes occur.

If you have any question, don´t hesitate to contact me!

Giambattista.

@Chantal, Thank you for your suggestion, how can I contact Dominique Madern...

Giambattista, we have only TA Q20 model DSC and we dont have nano DSC which is specific for Biomolecules. So I want to know whether I still can use TA Q20 or I have to look for Nano DSC in other labs. In Q20 the maximum sample volume, the pan can hold is 20ul. So I can only load less than 50 ug of protein sample. Can you please give some suggestion...

Up on my knowledge, for determining thermostability of an enzyme there is no need to do DSC and DSC can not give the enzyme activity with respect to a particular temperature. If you are determining the pyrolysis pattern of a peptide you can use DSC. To do the thermostability you should estimate the activity at different temperatures. The specific temperature at which the activity diminish is the maximum temperature (not Optimum) and you should select different temperatures according to that. If you need more information you can contact in e-mail.

Hello,

we have being working on the stability of several plant peroxidase using simultaneously complementary methods, such as DSC, CD, instrinsic fluorescence of proteins, enzyme activity measurements at different temperatures, pHs.

As examples:

Thermodynamic characterization of the palm tree Roystonea regia peroxidase stability. Biochimie 90, 1737- 1749 (2008)

Thermal stability of peroxidase from Chamaerops excelsa palm tree at pH 3,0. International Journal of Biological Macromolecules 44, 326-332 (2009)

Regards.

Use DSC by all means, however you may find Thermal Melt Circular Dichroism is informative also. Also using TMCD you can study reversibility of any unfolding/destabilisation trends observed +/-any analytes you wish (reducing agents etc).

There are many methods to determine the thermostability of proteins.

DSC is very good method as it gives a lot of information. you can also calculate rate constants of thermal denaturation + thermodynamic activation parameters. It gives you Melting temperature as well as enthalpy of denaturation (delta H).

Circular discroism (CD) in far-UV range gives you the melting temperature (Tm) of secondary structure whereas Near-UV CD gives you the Tm of tertiary structure.

Intrinsic Fluorescence of Trp or Tyr residues gives you the Tm of tertiary structure using very little protein whereas extrinsic fluorescence using ANS or nile red can also be used to determine thermostability.

My favorite method is TUG-PAGE (transverse urea gradient-PAGE) which can be set up easily in a lab and gives additional information about protein stability. I think I can not attach more than a single paper. If you contact me on [email protected], In can send you all the relavent info.

Here is the paper re TUG-Urea PAGE. Other papers are:

1.  Siddiqui, KS and Thomas, T., editors (2008) Adaptations in extremophilic proteins. Nova Science Publishers Inc., Hauppauge, NY,USA, ISBN 978-1-60456-019-0

2.  Cavicchioli, R., Curmi, PMG., Siddiqui, KS. and Thomas, T (2006) Proteins from Psychrophiles. In: Extremophiles - Methods in Microbiology Vol. 35. (eds. Rainey, FA. and Oren, A) Academic Press. 395-436 (invited)

3. Siddiqui, KS., Francisci, D. and Thomas, T (2006) Analysis of unfolding-refolding patterns and stabilities of proteins by transverse urea gradient gel electrophoresis (TUG-GE). In: Protein Structures. Methods in Protein Structure and Stability Analysis. (eds. Uversky VN. & Permyakov AE) Nova Science Publishers, Inc., Hauppauge, NY, USA (invited)

4.  Siddiqui KS and Cavicchioli R (2006) Cold adapted enzymes. Annual Review of Biochemistry. Vol. 75: 403-433 (invited)

5.  Siddiqui KS, Poljak A, De Francisci D, Guerriero G, Pilak O, Burg D, Raftery MJ, Parkin DM, Trewhella J, Cavicchioli R. (2010) A chemically modified α-amylase with a molten-globule state has entropically driven enhanced thermal stability. Protein Eng Des Sel 23(10):769-780.

6. Siddiqui KS, Parkin DM, Curmi PM, De Francisci D, Poljak A, Barrow K, Noble MH, Trewhella J, Cavicchioli R (2009). A novel approach for enhancing the catalytic efficiency of a protease at low temperature: reduction in substrate inhibition by chemical modification. Biotechnol Bioeng. 103(4):676-86.

7.  Siddiqui, KS., Poljak, A., Guilhaus, M., Francisci, D., Curmi, PMG., Feller, G., D’Amico, S., Gerday, C., Uversky, VN. and Cavicchioli, R (2006). The role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted -amylase from Pseudoalteramonas haloplanktis. Proteins: Struct. Func. Bioinformatics. 64(2):486-501

8.  Siddiqui, KS., Poljak, A., Guilhaus, M., Feller, G., D’Amico, S., Gerday, C. and Cavicchioli, R. (2005b) The role of disulphide-bridges in the activity and stability of a cold-active -amylase. J. Bacteriol. 187 (17): 6206-6212

I hope these helps.

Hi,

Differential scanning fluorimetry (DSF) also knonw as Thermofluor, or thermal shift assay, is a great technique for medium/high throughput determination of protein thermostability. Usually low amounts of protein are required and it is a is very fast method: in ~1 hour one can get thermostability data for dozens of different conditions.

See:

J Biomol Screen. 2001 Dec;6(6):429-40.

High-density miniaturized thermal shift assays as a general strategy for drug discovery.

Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E, Carver T, Asel E, Springer BA, Lane P, Salemme FR.

http://jbx.sagepub.com/content/6/6/429.long

And our papers using DSF to study stability of metabolic enzymes and model proteins in different ionic liquids conditions:

Cofactors and metabolites as potential stabilizers of mitochondrial acyl-CoA dehydrogenases.

Lucas TG, Henriques BJ, Rodrigues JV, Bross P, Gregersen N, Gomes CM.

Biochim Biophys Acta. 2011 Dec;1812(12):1658-63.

http://linkinghub.elsevier.com/retrieve/pii/S0925-4439(11)00204-3

Protein stability in an ionic liquid milieu: on the use of differential scanning fluorimetry.

Rodrigues JV, Prosinecki V, Marrucho I, Rebelo LP, Gomes CM.

Phys Chem Chem Phys. 2011 Aug 14;13(30):13614-6.

http://www.ncbi.nlm.nih.gov/pubmed/21709866