title: Use of Differential Scanning Fluorimetry to Identify Nuclear Receptor Ligands.
abstract: Identification of small molecules that interact specifically with the ligand-binding domains (LBDs) of nuclear receptors (NRs) can be accomplished using a variety of methodologies. Here, we describe the use of differential scanning fluorimetry to identify these ligands, a technique that requires no modification or derivatization of either the protein or the ligand, and uses an instrument that is becoming increasingly affordable and common in modern molecular biology laboratories, the quantitative, or real-time, PCR machine. Upon being introduced to specific ligands, nuclear receptors undergo structural and dynamic changes that tend to increase molecular stability, which can be measured by the resistance of the protein to heat denaturation. Differential scanning fluorimetry (DSF) uses a dielectric sensitive fluorescent dye to measure the thermal denaturation, or "melting" point (Tm) of a protein under different conditions, in this case in the absence and presence of a candidate ligand. Using DSF, multiple candidates can be screened at once, in numbers corresponding to plate size of the instrument used (e.g., 96- or 384-well), allowing significant throughput if a modest library of compounds needs to be tested.
authors.affiliation: State University of New York at New Paltz, 1 Hawk Drive, New Paltz, NY, 12561, USA.
authors.forename: Kara A
authors.initials: KA
authors.lastname: DeSantis
authors.affiliation: State University of New York at New Paltz, 1 Hawk Drive, New Paltz, NY, 12561, USA. reinkinj@newpaltz.edu.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Reinking
mesh_terms: D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008024:Ligands; D011485:Protein Binding; D011489:Protein Denaturation; D000072417:Protein Domains; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear
keywords: Compound library screening; Fluorescent dye; apo-Ligand-binding domain