Application Notes

2 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. alters its photophysical properties [6]. By detecting changes in tryptophan fluorescence intensity and its emission peak shi , the transition of a protein from the folded to the unfolded state can be precisely recapitulated. This way, the melting temperature (T m ) and thermodynamic parameters can be determined [7]. Here we demonstrate the performance of the Prometheus in monitoring thermal unfolding of proteins in a formulation screening project. The Prometheus NT.48 can measure up to 48 samples in parallel, and uses high-precision capillaries which are filled with just 10 µl of sample. Using a detector which is specifically designed to monitor changes in the emission spectrum of tryptophan with maximal sensitivity and speed, highest data point density and precision is achieved. Proteins of the α-amylase family are well-established for the analysis of protein folding [8]. Most amylases share very similar tertiary structures, with three (β/α)- Figure 1: Structure of pig pancreatic α-amylase (PPA, green) and Aspergillus oryzae α-amylase (TAKA, blue). The red sphere represents a Ca 2+ -ion. barrel domains and at least one conserved Ca 2+ -binding site (Figure 1). At the same time however, they show an extremely broad range of melting temperatures (from 40 °C to 110 °C), which makes them perfect candidates for basic research on the determinants of thermal stability of proteins [9]. In addition to their value for basic medical research, amylases are commercially used in the production of ethanol in sugars in large- scale industries. In the present example, we investigated the thermal unfolding of α-amylase from mammals (Pig pancreatic α-amylase, PPA) and fungi (Aspergillus oryzae α-amylase, TAKA). We recapitulated the stabilizing effects of calcium ions on protein conformation, and finally performed formulation screens using different additives which improve thermal stability to different degrees. Results Monitoring and analyzing thermal unfolding using Prometheus Prometheus monitors the shi of intrinsic tryptophan fluorescence of proteins upon unfolding by detecting the fluorescence at an emission wavelength of 330 and 350 nm. For determination of the protein melting point (T m , where half of the protein is folded and the other half is unfolded), either the fluorescence change in one of the two channels can be used, or alternatively, the ratio of the fluorescence intensities (F350/F330) can be plotted. The latter approach is preferred for most proteins, since the fluorescence ratio monitors both, the change in tryptophan fluorescence intensity as well as a Pig pancreatic a-amylase (PPA) Aspergillus oryzae a-amylase (TAKA) Pig pancreatic α-amylase (PPA) Aspergillus orzae α-amylase (TAKA)

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