Application Notes

2 Here, we analyzed the thermostability of the wild- type DsLOV protein and two mutated forms, which possess altered dark recovery kinetics [unpublished data], using the Prometheus NT.48 instrument by NanoTemper Technologies. Thus, we demonstrate the performance of the Prometheus NT.48 to monitor thermal unfolding of proteins that do not contain any tryptophan residues. Results The Prometheus NT.48 is capable of monitoring changes in fluorescence upon thermal unfolding in samples lacking tryptophan residues, by detecting the fluorescence signals from tyrosine or phenylalanine residues. For determination of the protein melting point T m where half of the protein is unfolded, either the fluorescence change in one of the two channels, or alternatively, the ratio of the fluorescence intensities (F350/F330) can be plotted. In order to determine the detection limit of the Prometheus NT.48 for tyrosine residues, we used the wild-type DsLOV protein at different protein concentrations of 1 mg/ml, 100 µg/ml, 50 µg/ml, 10 µg/ml, and 1 µg/ml, respectively. Thermal unfolding was carried out at a heating rate of 1 °C/minute within a heating range from 20 °C to 95 °C. Figure 2 shows the change of the fluorescence ratio for the wild-type DsLOV protein plotted against temperature. The Prometheus NT.48 was able to determine the transition point of the wild- type DsLOV protein down to a tyrosine concentration of 50 µg/ml. Figure 2 Detection of tyrosine fluorescence. Representative thermal unfolding curves of the DsLOV-wildtype protein at different tyrosine concentrations (n = 3 measurements). In a second experiment we analyzed the influence of various mutations on the stability of the DsLOV protein. As seen in Figure 3, the two mutated DsLOV variants, DsLOVM1, and DsLOVM2, show altered thermal unfolding behavior, compared to the wild-type protein. While for the wild-type protein we measured a T m value of 63.5 °C, the stability of the mutant DsLOVM1 is slightly reduced to a T m of 62.0 °C. The DsLOVM2 mutation, however, leads to a significantly altered stability, with a measured T m value of 67.7 °C. Protein T m (°C) DsLOVWT 63.5 ± 0.12 DsLOVM1 62.0 ± 0.15 DsLOVM2 67.7 ± 0.10 Figure 3 Thermal stability of the DsLOV variants. Plot of F350/F330 fluorescence ratio versus temperature of wild-type DsLOV, DsLOVM1, and DsLOVM2 (n = 3 measurements). A protein concentration of 7 mg/ml protein was used for all three samples. Conclusion Our results show that the Prometheus NT.48 by NanoTemper Technologies is capable of monitoring fluorescence emission changes in samples without tryptophan residues, and thus can determine thermal stability of these proteins. Accordingly, we were able to analyze the influence of various mutations on the stability of DsLOV, a photoreceptor protein without any tryptophan residues. These results demonstrate the broad application range of the Prometheus NT.48, covering the analysis of changes in fluorescence emission of tryptophans and tyrosines. 1 mg/ml Protein 100 µg/ml Protein 50 µg/ml Protein 10 µg/ml Protein 1 µg /ml Protein

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