TY - JOUR

T1 - Global analysis of protein stability by temperature and chemical denaturation

AU - Hamborg, Louise

AU - Horsted, Emma Wenzel

AU - Johansson, Kristoffer Enøe

AU - Willemoës, Martin

AU - Lindorff-Larsen, Kresten

AU - Teilum, Kaare

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - Chemical denaturation

KW - Data analysis

KW - Protein stability

KW - Temperature denaturation

U2 - 10.1016/j.ab.2020.113863

DO - 10.1016/j.ab.2020.113863

M3 - Journal article

C2 - 32738214

AN - SCOPUS:85088916695

VL - 605

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

M1 - 113863

ER -