Global analysis of protein stability by temperature and chemical denaturation.
The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires preparation of several samples and only provides the free energy of folding when performed at a single temperature. The typical sample requirement is around 0.5-1 mg of protein. If the stability of many proteins or protein variants needs to be determined, substantial protein production may be needed. Here we have determined the stability of acyl-coenzyme A binding protein at pH 5.3 and chymotrypsin inhibitor 2 at pH 3 and pH 6.25 by combined temperature and denaturant unfolding. We used a setup where tryptophan fluorescence is measured in quartz capillaries where only 10 μl is needed. Temperature unfolding of a series of 15 samples at increasing denaturant concentrations provided accurate and precise thermodynamic parameters. We find that the number of samples may be further reduced and less than 10 μg of protein in total are needed for reliable stability measurements. For assessment of stability of protein purified in small scale e.g. in micro plate format, our method will be highly applicable. The routine for fitting the experimental data is made available as a python notebook.
['Hamborg|Louise|L|', 'Horsted|Emma Wenzel|EW|', 'Johansson|Kristoffer Enøe|KE|', 'Willemoës|Martin|M|', 'Lindorff-Larsen|Kresten|K|', 'Teilum|Kaare|K|']
D002352:Carrier Proteins D019791:Guanidine D007700:Kinetics D010455:Peptides D010940:Plant Proteins D011487:Protein Conformation D011489:Protein Denaturation D055550:Protein Stability D013816:Thermodynamics D014508:Urea
Protein stability Chemical denaturation Data analysis Temperature denaturation