Application Notes

2 Using the Prometheus NT.48 instrument, we could demonstrate that thermal stability of PA2949 strongly depends on the detergent type, and that enzyme activity directly correlated with the determined T m -values. Results Using the Prometheus NT.48 instrument we have monitored the changes in fluorescence upon thermal unfolding of PA2949 in the presence of four different detergents: anionic sodium dodecyl sulfate (SDS), zwitterionic 3-(N,N- dimethylpalmitylammonio)propanesulfonate (SB3- 16), and the neutral detergents n-octyl β-D- glucoside (OG) and n-dodecyl β-D-maltoside (DDM). All detergents were used at concentrations above the CMC (critical micelle concentration) which are necessary for stabilization of membrane proteins (Table 1). The ratio of fluorescence intensities at 350 nm and 330 nm as a function of temperature was used to determine the transition temperature, which can be interpreted as the melting temperature of PA2949 (Figure 2). Interestingly, SDS and SB3-16 caused immediate unfolding of PA2949 as demonstrated by the absence of an unfolding signal by nanoDSF. OG only slightly destabilized the enzyme, and DDM had no negative effect on PA2949 stability (Table 1). Figure 2: Thermal stability of PA2949 measured with the fluorescence-based nanoDSF method. The fluorescence ratio (F 350nm /F 330nm ) and the derivative of the fluorescence ratio (Δ(F 350nm /F 330nm )/ΔT) as a function of temperature are shown in the upper and lower diagram, respectively. Colored dashed lines indicate the melting temperatures (T m ) in the presence of different detergents. We further determined thermal unfolding of PA2949 using a classical biochemical method, which relies on measurement of the residual esterase activity after exposure of PA2949 to different temperatures. These results revealed a complete loss of PA2949 activity in SDS and SB3- 16, OG inactivated PA2949 only slightly and DDM did not cause any inactivation (Figure 3, Table 1). The comparison of thermal stability of PA2949 in detergents studied by fluorescence and enzymatic methods revealed the same correlation of melting temperatures, T m (DDM) > T m (OG) > T m (SDS) = T m (SB3-16). However, the absolute melting temperatures of PA2949 in buffer measured enzymatically (T m (buffer) = 52.1 °C) was approximately 8 °C lower than the one measured with the fluorescence method (T m (buffer) = 59.9 °C). Figure 3: Thermal stability of PA2949 measured by determination of enzymatic activity. The relative esterase activity and the derivative of esterase activity (ΔActivity/ΔT) as function of temperature are shown in the upper and lower diagram, respectively. 100 % activity corresponds to the activity of PA2949 in buffer at 30 °C. Colored dashed lines indicate the melting temperatures (T m ) in the presence of different detergents. Table 1: Comparison of melting temperatures of PA2949 measured with nanoDSF and enzymatic activity assay Condition CMC* T m (fluorescence) [°C] T m (enzymatic) [°C] Buffer - 59.9 ± 1.0 52.1 ± 0.7 DDM, 0.2 mM 0.15 mM 59.3 ± 0.1 52.8 ± 0.5 OG, 34.2 mM 25 mM 53.1 ± 0.4 43.4 ± 0.5 SDS, 1.5 mM 8 mM unfolded unfolded SB3-16, 10 mM 3 mM unfolded unfolded *CMC: the critical micelle concentration is defined as the concentration of detergent above which micelles form.

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