Global analysis of protein stability by temperature and chemical denaturation

Abstract

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.

Synergistic stabilization of a double mutant in CI2 from anin-celllibrary screen

“…CI2 is a highly stable protein with free energy for folding, ∆Gf = 31 kJ/mol (Hamborg et al, 2020;Itzhaki et al, 1995) and, as previously shown (Zutz et al, 2020), when wild-type CI2 is expressed in the bacterial sensor system (Figure 1a) only little GFP is produced ( Figure 1b). The dynamic range of the GFP signal towards discovering stabilized variants (i.e.…”

“…To measure the in vitro stability for folding we used our recently described combined twodimensional thermal and chemical protein unfolding assay, where the unfolding of the protein is followed by the change in intrinsic Trp fluorescence as the temperature is increased at multiple concentrations of denaturant ( Figure 1d) (Hamborg et al, 2020). The stabilities of the 27 CI2 variants cover a broad range from -7.4 kJ/mol to -38.5 kJ/mol including five variants that are more stable than the wild-type protein (Table 1 and Figure 1e).…”

“…The temperature was ramped from 15 to 95°C with a temperature increment of 1°C/min. Global analysis of temperature and solvent denaturation was performed as described (Hamborg et al, 2020).…”

Universal amyloidogenicity of patient-derived immunoglobulin light chains

“…These plots are automatically generated by the control software of the Prometheus Panta instrument. For the combined chemical and thermal unfolding experiments, the data set of each protein was globally fitted to the thermodynamic two-state model recently presented 47 . The global fits are shown in supplementary figure 7.…”

“…Interestingly, we find that significant aggregation is only observed in the absence of urea, as well as for the 1-2 lowest urea concentrations. This information allowed us to perform a global fit to a model of the temperature-dependent IgLC thermodynamic stability 47 , where we excluded the samples where aggregation was observed. Details of the modelling, which was performed directly on the fluorescence intensity rather than the ratio, can be found in the methods section.…”