Chemicals and Reagents Butyrolactone I and other compounds were provided by Dr. Methods 2.1. Chemicals and Reagents Butyrolactone I and other compounds were provided by Dr. Jongheon Shin (Seoul National University, Seoul, Republic of Korea). The extraction and isolation were conducted as previously reported . Butyrolactone I Appearance: Pale yellow amorphous solid, +95 (1.0, EtOH), FT-IR (KBr, cm?1): 3179, 1763; 1H-NMR (DMSO-= 10.52 (brs, 1H), 9.92 (s, 1H), 9.12 (s, 1H), 7.50 (d, = 8 Hz, 2H aromatic H), 6.88 (d, = 8 Hz, 2H aromatic H), 6.53 (d, = 7.5 Hz, 1H), 6.47 (dd, = 8 Hz, 2 Hz, 1H), 6.37 (m, 1H), 5.01 (t, = 7.1 Hz, 1H), 3.74 (s, 3H), 3.36 (m, 2H), 3.00 (t, = 7 Hz, 2H), 1.62 (s, 3H), 1.53 (s, 3H); 13C-NMR (DMSO-= 169.8, 167.9, 157.8, 153.7, 138.0, 131.3, 130.8, 128.7, 128.3, 126.4, 123.1, 122.3, 121.0, 115.7, 114.0, 84.7, 53.4, 38.0, 27.5, 25.4, 17.4; HR-ESI-MS: strain. The cells were grown at 37 in Luria Broth media containing 30 g/mL kanamycin and induced by 0.5 mM isopropyl 1-thio–d-galactopyranoside at an OD600 of 0.6 and then incubated for additional 20 h at 20 . The cells were harvested Parsaclisib by centrifugation at 6000 for 10 min and lysed by sonication in buffer A (20 mM Tris-HCl pH 8.5, 150 mM NaCl, 5 mM imidazole, 10% glycerol, and 1 mM TCEP) containing 1 mM phenylmethanesulfonylfluoride. The lysates were centrifuged at 35,000 for an hour and the supernatants were filtered with a 0.45 m syringe filter device (Sartorius, G?ttingen, Germany). For affinity chromatography, they were loaded onto 5 mL HiTrap chelating HP column (GE Healthcare, Chicago, IL, USA) that was charged with Ni2+ and equilibrated with buffer A. Upon eluting with linear gradient of buffer B (20 mM Tris-HCl pH 8.5, 150 mM NaCl, 300 mM imidazole, 10% glycerol, and 1 mM TCEP), PPAR LBD was Parsaclisib eluted at an imidazole concentration of 50C100 mM. After the eluted protein was desalted using HiPrep Desalting column 26/10 (GE Healthcare) to buffer C (20 mM Tris-HCl pH 8.5, 150 mM NaCl, 10% glycerol, and 1 mM TCEP), the protein was treated with thrombin (Sigma-Aldrich) for the cleavage of His6-tag at 1 unit/mg and incubated at 4 overnight. The His6-tag-cleaved PPAR LBD was purified by passing through the Ni2+ charged HiTrap chelating HP column (GE Healthcare) to remove His6-tag or uncleaved His6-tagged target proteins, followed by gel Parsaclisib filtration chromatography column, HiLoad 16/600 Superdex 200 pg (GE Healthcare), that was previously equilibrated with buffer C. For crystallization, the PPAR LBD was concentrated to 15.5 mg/mL using an Amicon Ultra-15 Centrifugal Filter Unit (Merck Millipore, Darmstadt, Germany). 2.6. Crystallization The ligand-free PPAR LBD crystals were grown by the sitting-drop vapor diffusion method at 22 by mixing 0.5 Rabbit polyclonal to DYKDDDDK Tag L each of the purified protein sample and a crystallization solution containing 1.4 M sodium citrate tribasic dihydrate (Hampton Research, Aliso Viejo, CA, USA) Parsaclisib and 0.1 M HEPES pH 7.5. The crystals suitable for data collection were grown in the presence of micro-seeds that were made from the initial crystals using Seed Bead Kits (Hampton Research) according to the manufacturers instructions. The cubic-shaped crystals with a dimension of approximately 0.2 mm 0.2 mm 0.2 mm were obtained within a few days. For butyrolactone I-bound PPAR LBD, butyrolactone I was completely dissolved in 100% DMSO at 100 mM concentration and was soaked into ligand-free PPAR LBD crystals with 1:5 molar ratio containing 1% (. The structures were refined by iterative manual buildings in  and  in the CCP4 program suite. All refinement steps were monitored using an Rfree value  based on the independent reflections and the reliability of refined models was evaluated using . The statistics of data collection and refinement are summarized in Table 1. Table 1 Statistics for the data collection and model refinement. = hi|I(h)iC|/hiI(h)i, where I(h) is the intensity of reflection h, h is.