Hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD) is a degenerative disorder caused by single substitutions in highly conserved residues of p97/VCP. structures of all p97/VCP proteins revealed a didispersed distribution of a predominant hexameric population and a minor population of large-diameter complexes. ATP binding significantly increased the abundance of large-diameter complexes for p97R155P and p97A232E, but not p97WT or the ATP-binding mutant p97K524A. Therefore, we propose that hIBMPFTD p97/VCP mutants p97R155P and p97A232E possess structural defects that may compromise the mechanism of p97/VCP activity within large multiprotein complexes. The ubiquitous strain. Overnight starter cultures were diluted 50-fold in 2 liters of 2 YT medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl) with 100 g/ml ampicillin and grown at 37C until they reached an optical density at 600 nm of 0.6. The cultures were subsequently induced with 300 M IPTG (isopropyl–d-thiogalactopyranoside) and grown for another 4 h. The cells were pelleted by centrifugation (4,000 for 20 min) and lysed in buffer A (20 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM MgCl2, 2.5 mM dithiothreitol [DTT], and 20 mM imidazole) supplemented with 1 mg/ml lysozyme and complete protease inhibitors (Roche, Mississauga, Canada). Following sonication, the lysates were cleared by centrifugation at 26,000 for 45 min. The supernatant was then loaded on a 5-ml Ni2+ affinity column (GE Healthcare Biosciences, Uppsala, Sweden) preequilibrated with 10 column volumes of buffer A. Following a first wash with 10 column volumes of buffer A, bound proteins were subjected to an additional wash with 10 column volumes of buffer B (20 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM MgCl2, 2.5 mM DTT, and 35 mM imidazole). The proteins were eluted with a 35 to 500 mM imidazole gradient over 5 column volumes. Peak fractions containing p97 were pooled, concentrated on an Amicon Ultra column (molecular mass cutoff, 15,000 kDa), and loaded on a precalibrated 100-ml Superose-6 gel filtration column (GE Healthcare Biosciences, Uppsala, Sweden). Size-fractionated proteins were eluted as 1-ml fractions in buffer C (20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2, and 2.5 mM DTT). All purifications were performed at 4C using the UKTA fast-performance liquid chromatography purification system (GE Healthcare Biosciences, Uppsala, Sweden). Protein concentrations were determined using the Bradford assay (Bio-Rad, Mississauga, Canada). Native gel electrophoresis. Ten micrograms of purified recombinant p97WT or Rabbit Polyclonal to FUK mutant variants was loaded on a 6% Tris-polyacrylamide gel (pH 8.0). Electrophoresis was performed at 4C using 200 V and ice-cold 1 Tris-glycine buffer (pH 8.0) to prevent heat-induced denaturation. Negative-staining EM. p97 proteins were diluted to a concentration of 50 g/ml in buffer containing 20 mM HEPES, pH 7.5, 150 Tianeptine sodium mM NaCl, 10 mM MgCl2, and 1 mM DTT in the presence or absence of 1 mM ATP (Sigma, Oakville, Ontario, Canada). For each protein, a 5-l sample was spotted on a glow-discharged carbon-coated copper grid. The grids were then stained for 60 s with 5 l of uranyl acetate and visualized using an FEI Tecnai 12 120-kV electron microscope. Digital images were collected with a Gatan 792 Bioscan 1,000 by 1,000 wide-angle multiscan charge-coupled device camera. Images were cropped at the level of the scale bar to Tianeptine sodium show 25% of the full image. Tianeptine sodium Dynamic-light-scattering (DLS) measurements. p97 protein samples were diluted to 100 g/ml in buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 1 mM DTT in the presence or absence of 2 mM ATP. Measurements were performed using the Zetasizer Nano ZS (Malvern) at 20C. ATPase activity assays. p97 ATPase activity was assessed using the EnzCheck phosphate assay kit (Molecular Probes, OR) as previously described (6). This assay is based on the inorganic-phosphate-dependent conversion of 2-amino-6-mercapto-7-methylpurine riboside to ribose-1-phosphate and 2-amino-6-mercapto-7-methylpurine, Tianeptine sodium which results in a shift of the maximum absorbance from 330 nm to 360 nm (29). For assessment of p97 ATPase activity, 50 nM of p97 (based on the 600-kDa hexamer) was incubated in 100 l reaction buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, and 10 mM MgCl2) containing 200 M of ATP. Parallel reactions were set without the addition of ATP to correct for background absorbance values. The absorbance at 360 nm was then measured at 20-s intervals for a total of 20 min at the physiological temperature.