Application Notes

1 Thermal Stability of Membrane-bound Proteins Application Note NT-PR-012 nanoDSF Thermal Unfolding Analysis of a Membrane-bound Esterase in Various Detergents Florian Bleffert 1 , Vinko Misetic 2 , Moran Jerabek 2 , Karl-Erich Jaeger 1,3 & Filip Kovacic 1 1 Institute of Molecular Enzyme Technology, Forschungszentrum Juelich, 52426 Juelich, Germany 2 NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich, Germany 3 Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Juelich GmbH, D-52426 Juelich, Germany Abstract nanoDSF, the miniaturized differential scanning fluorimetry technology, is a revolutionary method to determine the thermostability of proteins by following changes in their intrinsic fluorescence. In this comparative study, the Prometheus NT.48 was used to determine the thermal stability of the membrane esterase PA2949 from Pseudomonas aeruginosa in presence of various detergents. The detergent type strongly affected enzyme thermal stability, which moreover correlated with enzyme activity. Thus, the Prometheus NT.48 can be used not only to rapidly screen for optimal purification conditions for enzymes, but also to evaluate enzyme activities based on their conformational stability in presence of detergents. Introduction Intrinsic protein fluorescence can mainly be attributed to the fluorescence of tryptophan (Trp) residues which is strongly sensitive to the polarity of the Trp micro-environment. Trp fluorescence is excited at 280 nm and emission occurs at 330 nm for Trp in non-polar environment and at 350 nm for Trp in polar environment [1]. Upon protein unfolding, Trp residues which are normally hidden in the protein hydrophobic core or in the detergent micelle get exposed to water resulting in a decrease of their fluorescence intensity and a shift of their emission maximum to longer wavelengths. Thus, by measuring the changes in Trp fluorescence intensity, the melting temperature (T m ) of proteins can be determined in a dye-free approach [2]. This approach is used by nanoDSF and has already been successfully applied for detergent-solubilized membrane proteins. This protein class is typically difficult to analyze with other techniques such as Thermofluor, since the hydrophobicity of the detergent micelles prevents the use of unfolding-reporting dyes such as Sypro- Orange. Also, differential scanning calorimetry is very sensitive towards different detergent types, limiting its applicability for experiments with membrane proteins. Since detergents are indispensable for successful purification and storage of membrane proteins, the screening for the optimal detergent is crucial, but is often still performed using a sub-optimal trial-and-error principle which is time consuming, unproductive and expensive. Here, we analyzed the thermostability of a membrane-bound esterase (PA2949) from Pseudomonas aeruginosa PA01 [3]. PA2949 hydrolyses the racemic methyl ester of β- acetylthioisobutyrate to produce the (D)-β- acetylthioisobutyric acid (DAT). DAT is a valuable key intermediate for the synthesis of captopril (Figure 1), a drug used for treatment of hypertension [4]. Figure 1: Chemical structure of captopril with (D)-β- thioisobutyrate group produced by PA2949 depicted in red.

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