multi-angle laser light scattering (mals) module down heleos ii  (Waters Corporation)

Journal: bioRxiv

Article Title: Periphilin self-association underpins epigenetic silencing by the HUSH complex

doi: 10.1101/2019.12.18.881300

Figure Lengend Snippet: Periphilin and TASOR form a 2:1 complex required for HUSH function. ( A ) Crystal structure of the Periphilin-TASOR core complex. The Periphilin fragment (residues 292-367, light/dark grey) forms a homodimer of helical hairpins. The TASOR fragment (residues 1014-1095, rainbow colors) wraps around the Periphilin dimer, adding an α -helix to each Periphilin hairpin to form two helical coiled coils. Insets show close-up views of the Periphilin-TASOR interfaces (“i”, “iii”) and the Periphilin dimer interface (“ii”). Residues forming key contacts and mutations designed to disrupt Periphilin-TASOR complex formation are labeled. ( B ) SEC-MALS of Periphilin-TASOR core complex. The molecular weight calculated from light scattering data is consistent with a 2:1 complex in solution. ( C ) Repression of a lentiviral GFP reporter in Periphilin KO cells complemented with Periphilin mutants designed to inhibit Periphilin-TASOR complex assembly. Reporter expression was monitored over 21 days by flow cytometry. The log 10 (GFP fluorescence) data for live cells were converted to percent repression activity with wild-type HeLa set at 100% and Periphilin KO cells set a 0% repression (see Methods). ( D ) Immunofluorescence microscopy of Periphilin KO cells transduced with Periphilin mutants affecting Periphilin-TASOR complex assembly. Cells were fixed 4 days post-transduction and stained with anti-Periphilin antibody (magenta) and DAPI (grey, insets). Scale bar, 10 µm.

Article Snippet: The SEC system was coupled to a multi-angle light scattering (MALS) module (DAWN-8+, Wyatt Technology).

Techniques: Labeling, Molecular Weight, Expressing, Flow Cytometry, Fluorescence, Activity Assay, Immunofluorescence, Microscopy, Transduction, Staining

Journal: Nature Communications

Article Title: Neuropathic MORC2 mutations perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms

doi: 10.1038/s41467-018-03045-x

Figure Lengend Snippet: MORC2 is a GHKL-type ATPase. a Domain organization of human MORC2. The GHKL ATP binding domain and the transducer-like domain (trans.) together form the ATPase module, as marked. CC indicates a predicted coiled coil; CW indicates a CW-type zinc finger domain; TCD indicates a predicted tudor-chromodomain. b Fitted T m s derived from differential scanning fluorimetry (DSF) for several MORC2 variants at 5 µM, in the absence (solid bar) and presence (striped bar) of 2 mM Mg-AMPPNP. This non-hydrolysable ATP analog significantly increases the thermal stability of wild-type (WT) MORC2(1–603), while the N39A point mutant abrogates binding and WT MORC2(1–282) is stabilized to a much smaller extent than WT MORC2(1–603). Quoted T m values are an average of at least two replicates; note that the deviation between these measurements was <0.2 °C in all cases. c Portions of overlaid SEC-MALS UV traces for 40 µM WT MORC2(1–603) in the absence (solid line) and presence (dashed line) of 2 mM Mg-AMPPNP. The MALS data across the center of the major peaks in each case are shown on the right-hand axis, and are consistent with monomeric (expected mass: 70 kDa) and dimeric (expected mass: 140 kDa) species for the apo and AMPPNP-bound protein, respectively. Also shown are the fitted hydrodynamic radii obtained from QELS analysis of the peaks. The peak at 48 min in the AMPPNP-treated trace is the elution of excess (unbound) nucleotide. d Rate of ATP hydrolysis by wild-type (WT) and N39A MORC2(1–603) variants at 37 °C in the presence of 7.5 mM ATP, measured using an NADH-coupled continuous assay. Error bars represent standard deviation between measurements; n = 8 (WT), n = 7 (N39A). e Steady-state ATPase activity of 4 µM WT MORC2(1–603) at 37 °C fitted to a model of Michaelis–Menten kinetics

Article Snippet: The SEC system was coupled to both MALS and QELS modules (Wyatt Technology).

Techniques: Binding Assay, Derivative Assay, Mutagenesis, Standard Deviation, Activity Assay