期刊
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
卷 181, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.jpba.2019.113065
关键词
Protein thermal shift assay; Thermostability; Melting point; Guanylyl cyclase; sGC activators; Nitric oxide
资金
- European Community Seventh Framework Programme (FP7/2007-2013) under BioStruct-X [283570]
Thermal shift assay is a fluorescence dye based biochemical method to determine the melting point of a protein. It can be used to investigate the ligand-induced stabilization of proteins and helps to increase the likelihood of crystallization in biological samples. Dimeric proteins like soluble guanylyl cyclase (sGC) have specific structural and functional properties which may pose a challenge in thermal shift measurements. In this paper, thermal shift assay was used to examine ligand-induced thermostabilization of the dimeric heme-containing protein soluble guanylyl cyclase. Adjustment of the parameters buffer solution, pH, protein / dye ratio and protein amount per well yielded a one-phase melting curve of sGC with a sharp transition and high reproducibility. We found that thermal shift measurement is not affected by heme state or heme content of the enzyme preparation. We used the method to investigate the thermostabilization of sGC induced by the heme-mimetic activator drugs cinaciguat, BAY 60-2770 and BR 11257 in combination with non-hydrolyzable nucleotides. Measurements with the dicarboxylic drugs cinaciguat and BAY 60-2770 yielded steep melting curves with high amplitudes. In contrast, in the presence of the monocarboxylic sGC activator BR 11257, melting curves appear flattened in the dye-based measurements. In the present paper, we show that activity-based thermostability measurements are superior to dye-based measurements in detecting the thermostabilizing influence of sGC activator drugs. (C) 2019 Elsevier B.V. All rights reserved.
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