I am working with a protein that precipitates at 45 degree Celsius and above. I want to do thermodynamic characterization of the protein. Specifically I am interested in estimation of the heat capacity change upon unfolding. I cannot use ITC, DSC and thermal unfolding. I would like to know other experimental options to estimate heat capacity change upon unfolding of protein.
You might also look at different ways of characterizing thermal stability, like kinetics of unfolding; or, consider a Lumry-Eyring type of model to analyze your data. Here are a few references that may or may not be helpful.
1. Camarillo-Cadena, M.; Garza-Ramos, G.; Peimbert, M.; Perez-Hernandez, G.; Zubillaga, R. A. Thermal Denaturation of beta-Glucosidase B from Paenibacillus polymyxa Proceeds Through a Lumry-Eyring Mechanism. Protein J. 2011, 30 (5), 318-323.
2. Guzzi, R.; LaRosa, C.; Grasso, D.; Milardi, D.; Sportelli, L. Experimental model for the thermal denaturation of azurin: A kinetic study. Biophys. Chem. 1996, 60 (1-2), 29-38.
3. Sanchezruiz, J. M. THEORETICAL-ANALYSIS OF LUMRY-EYRING MODELS IN DIFFERENTIAL SCANNING CALORIMETRY. Biophys. J. 1992, 61 (4), 921-935.
Does your protein precipitate before or after unfolding?
In order to experimentally determine the heat capacity change upon denaturation you have to be able to observe the unfolding transition. If your protein precipitates before it denatures you should try to change solution conditions, pH, ionic strength, etc.
If you do observe the unfolding transition, than you can determine dCp with any method you want. It just takes more experiments than in DSC and the obtained value is less reliable.
Hi Nilesh,
Why do you say you cannot use DSC? You probably can. If you change the pH of the buffer to the acidic one (pH 2-3-4) the aggregation problem often disappears. You will probably get different value for the heat capacity change compared to the neutral pH (lower stability at lower pH), but sometimes it's accepted.
Another possibility is that your protein of interest is unfolded already at 45 degrees.
Can you give some information about your protein, its source and the buffer you are using now.
There are few tips based on my experience:
1. Using 0.5 to 1.0 M sulfobetaine or MOPS buffer can prevent aggregation before unfolding. look for Feller, G paper about the use of this detergent to avoid precipitation in DSC experiments.
2. Use as less protein concentration as possible. May be 100 to 200 ug/ml.
3. If you use latest nano-capillary DSC rather than old VP-DSC, aggregation before unfolding can be avoided. I recently started using nano-capillary DSC and found that it is much better as you can use lower protein concentrations and its design avoids aggregation/ppt.
4. As suggested above, pH has dramatic effect on aggregation. Use pH away from the pI of protein.
I hope it helps.
since aggregation is dependent on concentration i will use low concentration. for many proteins i get decent CD signal even for 200 nM protein.you have to use a 1cm pathlength cuvette for that. you can try temperature induced denaturation experiment by CD.
You might also look at different ways of characterizing thermal stability, like kinetics of unfolding; or, consider a Lumry-Eyring type of model to analyze your data. Here are a few references that may or may not be helpful.
1. Camarillo-Cadena, M.; Garza-Ramos, G.; Peimbert, M.; Perez-Hernandez, G.; Zubillaga, R. A. Thermal Denaturation of beta-Glucosidase B from Paenibacillus polymyxa Proceeds Through a Lumry-Eyring Mechanism. Protein J. 2011, 30 (5), 318-323.
2. Guzzi, R.; LaRosa, C.; Grasso, D.; Milardi, D.; Sportelli, L. Experimental model for the thermal denaturation of azurin: A kinetic study. Biophys. Chem. 1996, 60 (1-2), 29-38.
3. Sanchezruiz, J. M. THEORETICAL-ANALYSIS OF LUMRY-EYRING MODELS IN DIFFERENTIAL SCANNING CALORIMETRY. Biophys. J. 1992, 61 (4), 921-935.
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