Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery
| Autor: | Jeroen Molenaar, Alexander Fish, Sridhar Sreeramulu, Harald Schwalbe, Nadide Altincekic, Misbha Ud Din Ahmad, Christian Richter, Hans Wienk, Anastassis Perrakis |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Thermal shift assay
General Immunology and Microbiology Chemistry Fragment (computer graphics) General Chemical Engineering General Neuroscience Fragment-based lead discovery Temperature Proteins Context (language use) Nano differential scanning fluorimetry Fluorescence spectroscopy General Biochemistry Genetics and Molecular Biology Structural biology Fluorometry Target protein Biological system |
| Zdroj: | Journal of Visualized Experiments |
| ISSN: | 1940-087X |
| DOI: | 10.3791/62469-v |
| Popis: | Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments. |
| Databáze: | OpenAIRE |
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