Protein was expressed in E. coli BL21(DE3) cells and purified as described (18 (link)) with slight modifications. Briefly, cells were harvested and lysed in 50 mM Tris (pH 8.0), 5% glycerol, 0.25 % (w/v) 1-O-n-octyl-β-D -glucopyranoside, 1 mM β-mercaptoethanol (BME), 10 μg/mL phenylmethanesulfonyl fluoride, and 1 μg/mL Nα-p-tosyl-L -arginine methyl ester hydrochloride. The soluble portion of the lysate was loaded onto either nickel-nitrilotriacetic acid or TALON affinity resin (preequilibrated with 50 mM Tris (pH 8.0), 500 mM KCl, 5% glycerol, 10 mM imidazole, and 1 mM BME) and eluted with two steps of 50 mM and 250 mM imidazole. Protein fractions were pooled, dialyzed into the same buffer with no salt, and loaded onto a GE HiTrap Q HP column. Protein was eluted with a stepwise gradient from 0 to 500 mM KCl, and protein fractions were then concentrated to < 5 mL and loaded onto a Superdex 26/60 Amersham column preequilibrated in Superdex buffer (50 mM Tris (pH 8.0), 150 mM KCl, 5% glycerol, and 1 mM dithiothreitol) to yield > 95 % pure protein.
Metal-free HDAC8 was prepared as previously described (23 (link)). All plastics were presoaked in 1 mM EDTA followed by thorough rinsing with Milli-Q ddH2O. All pipette tips and sample tubes were certified trace metal free. For Co2+-HDAC8 activity measurements, metal-free protein was incubated on ice with equimolar Co2+(Aldrich, 99.998% trace metals basis).
Activity measurements for HDAC8 variants were performed as previously described for the wild-type enzyme (23 (link)) using the commercially available Fluor de Lys kit (BIOMOL) with Fluor de Lys HDAC8 substrate. Because of the increased catalytic efficiency of Co2+-HDAC8 in comparison with Zn2+-HDAC8 (23 (link)), Co2+-substituted D101 and H143 variants were prepared to ensure the measurement of residual catalytic activity. Accordingly, HDAC8 variants that were inactive with Co2+ were also inactive with Zn2+. Assays of catalytically active variants were performed in triplicate or quadruplicate. The IC50 assay mixture used to study HDAC8-APHA complexation contained 50 μM fluorogenic peptide substrate, 0.5 μM HDAC8, 25 mM Tris (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 0.1–100 μM APHA. Reactions were incubated at 37 degrees for 30 minutes, quenched with stop solution (Developer II (BIOMOL) and 1 μM TSA), and tested for deacetylated product using a fluorescence plate reader (Fluoroskan II) (ex = 355 nm, em = 460 nm).
Metal-free HDAC8 was prepared as previously described (23 (link)). All plastics were presoaked in 1 mM EDTA followed by thorough rinsing with Milli-Q ddH2O. All pipette tips and sample tubes were certified trace metal free. For Co2+-HDAC8 activity measurements, metal-free protein was incubated on ice with equimolar Co2+(Aldrich, 99.998% trace metals basis).
Activity measurements for HDAC8 variants were performed as previously described for the wild-type enzyme (23 (link)) using the commercially available Fluor de Lys kit (BIOMOL) with Fluor de Lys HDAC8 substrate. Because of the increased catalytic efficiency of Co2+-HDAC8 in comparison with Zn2+-HDAC8 (23 (link)), Co2+-substituted D101 and H143 variants were prepared to ensure the measurement of residual catalytic activity. Accordingly, HDAC8 variants that were inactive with Co2+ were also inactive with Zn2+. Assays of catalytically active variants were performed in triplicate or quadruplicate. The IC50 assay mixture used to study HDAC8-APHA complexation contained 50 μM fluorogenic peptide substrate, 0.5 μM HDAC8, 25 mM Tris (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 0.1–100 μM APHA. Reactions were incubated at 37 degrees for 30 minutes, quenched with stop solution (Developer II (BIOMOL) and 1 μM TSA), and tested for deacetylated product using a fluorescence plate reader (Fluoroskan II) (ex = 355 nm, em = 460 nm).
