Journal
ANALYTICAL BIOCHEMISTRY
Volume 605, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2020.113863
Keywords
Protein stability; Chemical denaturation; Temperature denaturation; Data analysis
Funding
- Novo Nordisk Foundation [NNF15OC0016360]
- Novo Nordisk Fonden [NNF18OC0033950, NNF15OC0016360] Funding Source: researchfish
Ask authors/readers for more resources
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 chyrnotrypsin 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 mu l 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 mu g 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available