Application Notes

1 2 A P P L I C A T I O N N O T E PART 2: THERMAL UNFOLDING ANALYSIS Following the isothermal DLS measurements, a thermal ramp is applied to the same 10 µL of sample loaded in the capillary. nanoDSF uses the intrinsic fluorescence of the protein to gain insight into its conformational stability, while the simultaneous turbidity and DLS measurements evaluate the overall colloidal stability of the membrane protein-nanodisc complexes. The first key parameter that was evaluated is the temperature of the onset of cumulant radius (T onset r H ). This is the temperature at which a size change for the population of nanodiscs is observed, indicating that the particles start losing structural integrity. Accordingly, this parameter was selected as the criterion for evaluating the thermal stability of the copolymer nanodiscs in their entirety. The respective values are presented in Table 2, with the samples with the three highest values highlighted in red for each membrane protein. It is not sufficient to consider solely the thermal stability of the nanodisc structure. The copoly- mer must also be selected based on the thermal stability of the protein embedded in the nano- discs. NanoDSF measures the intrinsic fluorescence intensities of proteins at two wavelengths – 350 nm and 330 nm – with high precision to gain insight into the unfolding of proteins. When a protein unfolds in solution, a change in the environment of the tryptophan and tyrosine residues will result in a corresponding alteration in the fluorescent emission spectrum. This is represented by the fluorescence ratio (350 nm/330 nm) changes along a thermal ramp. For a membrane protein stabilized by a synthet- ic copolymer nanodisc, the nanoDSF data does not necessarily exhibit a distinct unfolding tran- sition in the fluorescence ratio, in contrast to the membrane protein solubilized in solution by de- tergent. This could be explained by two reasons. First, the structure of the nanodisc is o en more thermally stable than the protein, which allows for protein unfolding to occur with minimal con- formational changes while embedded in the nanodisc. Second, the high lipid content of the nanodisc limits the changes in hydrophobici- ty upon unfolding, thereby limiting the chang- es observed in the emission spectrum. Conse- quently, the unfolding can be observed in some cases by measuring the fluorescence intensity at 350 nm (as demonstrated by WbaP in Table 2 and Figure 3).

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