Use of differential scanning fluorimetry as a high-throughput assay to Article

Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands - Article Example As a partial solution to this problem differential scanning fluorimetry (DSF) is normally used. This technique allows for the identification of conditions that enhance the stability of proteins. DSF is used for this process as it allows possible ligands that have not been modified to be screened as 10 µL reactions in 96-well format against partially purified protein, revealing particular interactors. The researchers aim to find out whether differential scanning fluorimetry permits screening without modification of either the protein and/or the ligand in a high-throughput fashion using a commercially-available nuclear receptor ligand candidate chemical library. The researchers attempt to identify interactors of the human estrogen receptor ? ligand binding domain (ER? LBD). They will also investigate whether compounds that come into contact with the ligand stabilize the protein and lead to an increase of the thermal denaturation point. This process will be monitored using the SYPRO d ye which is able to detect even slight environmental changes. BACKGROUND INFORMATION Nuclear receptors (NRs) are transcription factors that exist in all metazoa that control cellular activities. Incidentally, the activities of NRs are regulated by interactions that occur between small-molecule ligands and a structurally-conserved ligand-binding domain (LBD) of the NR. This interaction induce structural changes resulting in new interaction surfaces for the recruitment of transcriptional co-activators and/or co-repressors. A lot of attention has been focused on the identification of ligands that bind to NRs and this is pushed by two main factors: i. Identification of natural ligands of a given NR is important in comprehending the role of the NR within the organism, particularly when the ligand of the NR is not clearly known or understood; ii. Identification of the ligands is important in the development of drugs and has led to a rise of different techniques each with known strengths a nd weaknesses (DeSantis et al. 2). Differential scanning technologies permit identification of conditions that improve the stability of proteins. The conversion from native to denatured protein is measured in relation to temperature changes. Two differential scanning techniques that can be used are scattering (Differential Scanning Light Scattering) or the fluorescence of an environmentally-sensitive dye (Differential Scanning Fluorescence – DSF). The techniques allow for the variation of a number of variables such as pH, salt, addition of small molecules (Schulman and Heyman 642). Cell conditions that result into a shift of proteins to higher denaturation temperature are identified as â€Å"stabilizing† conditions. This knowledge has been used to create conditions that are optimal for crystal growth (Vedadi et al., 2006). Other studies have used this technique to establish that peptides can shift the denaturation point of proteins to a higher temperature. This paper a ttempts to show that DSF can be used to successfully identify known interactors of the estrogen receptor ? (ER?) from a commercially available compound library. MATERIALS AND METHODS i. Reagents and instruments His6-hER? LDB (302-552) expression vector Terrific Broth IPTG EDTA-Free Protease Inhibitor Tablets Branson Sonifier 450 Ni-NTA Superflow ?-estradiol pET15b Sypro Orange, 5000X CFX96 Real-Time PCR System Nuclear Receptor Ligand Library BML-2802 ii. Methods The first step in this study was the