Application Notes

nanoDSF thermal unfolding analysis of proteins without tryptophan residues

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1 Thermal Unfolding Application Note NT-PR-007 nanoDSF Thermal Unfolding Analysis of Proteins Without Tryptophan Residues Vinko Misetic 1 , Oliver Reiners 2 , Ulrich Krauss 2 , Karl-Erich-Jaeger 2 1 NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich, Germany 2 Institute of Molecular Enzyme Technology, Forschungszentrum Juelich, 52426 Juelich, Germany Abstract The nanoDSF (minituarized differential scanning fluorimetry) technology is a powerful method to determine the thermal and chemical stability of proteins by following changes in fluorescence, where the amino acid tryptophan serves as the main source for the fluorescence signal. We analyzed the stability of the light-oxygen- voltage (LOV) protein of the marine phototrophic α-proteobacterium Dinoroseo- bacter shibae (DsLOV), which does not contain any tryptophan residues. Further, we examined the impact of various mutations on DsLOV protein stability. This study demonstrates the ability of the Prometheus NT.48 to analyze protein stability in samples that do not contain any tryptophan residues. Introduction Fluorescence of the amino acid tryptophan, which is usually located within the hydrophobic core of a protein, is highly sensitive to changes in its immediate environment. Upon thermal or chemical unfolding, it gets exposed to the aqueous solvent phase, leading to a change in its photophysical properties. By detecting the changes in tryptophan fluorescence intensity and the emission peak shift, the melting temperature T m of proteins can be determined in a dye-free approach [1]. The indole group of tryptophan is the dominant fluorophore in proteins. It has a significantly stronger fluorescence and a higher quantum yield as compared to the other aromatic amino acids, namely tyrosine and phenylalanine [1]. Blue-light photoreceptors containing light-oxygen- voltage (LOV) domains regulate a myriad of different physiological responses in both eukaryotes and prokaryotes [2], by utilizing a conserved photochemistry [3]. The recently identified short LOV protein DsLOV, of the marine phototrophic α-proteobacterium Dinoroseobacter shibae, is involved in the upregulation of photopigment synthesis in the absence of blue light in the organism [5]. As a short LOV protein, DsLOV consists of a LOV sensory domain but lacks any fused effector domain [5]. Mutations within the LOV domain can lead to altered dark recovery kinetics [4]. The DsLOV protein does not contain any tryptophan residue, but possesses five tyrosine and seven phenylalanine residues, which contribute to the intrinsic fluorescence of the protein (Figure 1). Figure 1 X-ray structure of the DsLOV protein (PDB-ID: 4KUK). Side chains of aromatic amino acids are highlighted as spheres with the respective carbon atoms in green (Phe) or yellow (Tyr). Nitrogen and oxygen atoms are shown in blue and red, respectively. The flavin chromophore of DsLOV is shown in stick representation with carbon atoms in light blue, nitrogen atoms in dark blue and oxygen in red.

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