Fluoromax 3 spectrometer

Affinities of hAgo2 or MjAgo for guide and target substrates were measured in hAgo2 binding buffer (10 mM Tris pH 7.5, 100 mM KCl) or MjAgo binding buffer (10 mM Tris pH 7.5, 100 mM KCl and 1 mM MgCl₂) using a 700 μl quartz cuvette at a constant temperature of 25°C. Either 20 nM of FAM-labelled guide strand or binary complexes (500 nM of one of the Ago proteins and 20 nM of a FAM-labelled guide strand) were titrated with increasing concentrations of Ago or target strand, respectively. The fluorophore was excited at 492 nm and the change of fluorescence upon binding was recorded at 516 nm with slits set to 1 nm using a Fluoromax-3 spectrometer (Horiba Jobin Yvon). Data were mathematically evaluated with GraFit 5.2 (Erithacus Software) using a quadratic equation (Fluorescence = Fmax − ((c[guide] + L + Kd) − sqrt((sqr(c[guide] + L + Kd)) − 4*c[guide]*L))*(Fmax − Fmin)/((2*c[guide])), where Fmax is the maximum fluorescence, Fmin is the minimum fluorescence, c[guide] is the concentration of substrate, L is the concentration of the titration partner, and Kd is the equilibrium dissociation constant).

Samples for ThT fluorescence assay were prepared with pelleted TDP-43 filaments at the concentration of 250 µg ml⁻¹ and αSyn fibrils at 150 µg ml⁻¹ were mixed with 20 µM ThT in 30 mM Tris and 100 mM NaCl at pH 7.4. All fluorescence spectra obtained were an accumulation of three curves, recorded using FluoroMax3 spectrometer (Horiba) at room temperature using an excitation wavelength of 450 nm with a slit width adjusted to 1 nm and emission wavelength from 460 to 650 nm with a slit width at 5 nm. For ThS fluorescence, samples were mixed with 20 mM ThS and measured using the same settings as described above.

Steady-state fluorescent emission was recorded for 1 μM Cyp40 in the apo form and after the addition of 250 μM SRMEEVD (Hsp90 peptide) between 15°C and 45°C at 5°C intervals in 50 mM Hepes, pH 8; 100 mM sodium chloride; 1 mM DTT (Fluoromax-3 spectrometer, Horiba). Tryptophan was selectively excited at 295 nm (5 nm band pass excitation and emission) and emission spectra recorded from 310 to 400 nm, 1 nm interval; integration time 1 s; scans were repeated in triplicate, mean spectra are presented. Protein scans were blanked by subtracting a spectrum of a similar solution without protein at each temperature.