Bis ans

Fluorescence measurements were carried out on a Hitachi F-7000 Fluorescence Spectrophotometer. Emission spectra of α-Clu or β-Clu (0.2 mg/ml in PBS, pH 7.4) were recorded with the excitation wavelength set at 295 nm. The hydrophobic probe, 4,4′-Dianilino-1,1′-Binaphthyl-5,5′-Disulfonic Acid (bis-ANS) (Molecular Probes, USA), was used at a final concentration of 10 μM for bis-ANS-binding studies. The samples were excited at 390 nm. All emission spectra were recorded with the excitation and emission band passes set at 2.5 nm. Buffer spectra were recorded and subtracted from protein spectra to obtain the final measurements. All spectra were recorded at a temperature of 25 °C.

All experiments were performed on a CaryEclipse (Varian) spectrofluorometer in a temperature-controlled cell (30°C) in phosphate buffer (50 mM KH₂PO₄, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 15 mM ME). Recombinant proteins were titrated by fluorescent probe bis-ANS (Molecular Probes). Protein samples (0.05 mg/ml, or 2.3 μM per HspB8 monomer) were titrated by a stock solution of bis-ANS, so that the final concentration of the probe was in the range of 1–15 μM. Fluorescence was excited either at 295 (to excite Trp and bis-ANS) or 385 (to excite only bis-ANS) nm (slit width 5 nm) and emission spectra were recorded in the range of 305–590 nm or 400–590 nm (slit width 5 nm).

Amicon ultra centrifugal filters were obtained from Merck-Millipore (Merck Millipore, Germany). Bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium Salt) and iodoacetamide-fluorescein (IAF) were from Molecular Probe (Life Technologies do Brasil Ltda). Bromophenol blue, tetramethylethylenediamine (TEMED), molecular weight marker Kaleidoscope, glycerol, glycine, acrylamide were obtained from Bio-Rad Laboratories (CA, USA). Acids and solvents (HPLC grade) were from J.T. Baker (Avantor Performance Materials, Mexico). All other reagents were from Sigma (St. Louis, MO). All aqueous solutions were prepared with ultrapure water purified by a Direct-Q3 system (Merck Millipore, Germany) and treated with Chelex 100 before use.

BisANS (4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) powder (Sigma-Aldrich) was dissolved in MilliQ water and its concentration was determined spectrophotometrically using NanoDrop 2000/2000c Spectrophotometer (ThermoFisher) based on BisANS absorbance at 385 nm and known extinction coefficient (ε_385nm,water = 16,790 L mol⁻¹ cm⁻¹) (Sharma et al., 1998 (link)). Fluorescence measurements were performed at 37°C on a FluoroLog-3 Modular Spectrofluorometer (HORIBA Jobin Yvon) in a 10.00-mm Quartz glass cuvette (High Precision Cell, Hellma Analytics) with a magnetic stirrer. 1 μM 14-3-3ζ proteins in 1 mL of PBS were titrated with BisANS to final concentration 1–30 μM. After each addition of BisANS, the system was equilibrated for 6 min and then fluorescence of BisANS was excited at 385 nm and detected in the range 400–700 nm with entrance/intermediate/exit slit widths set to 1.5 mm. BisANS fluorescence intensity at 495 nm was used for assessment of protein hydrophobicity. Between measurements of different proteins, the cuvette was cleaned with 3 M HNO₃ for 30 min while stirring to remove all potential contaminants.

Three microliters of either α-Syn or tau aggregates (0.5 and 0.6 μg/ μl, respectively) and 247 μl of 10 μM bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt, Invitrogen) prepared in 100 mM glycine-NaOH buffer (pH 7.4) were added to the wells of 96-well clear-bottomed black plates. Each condition was performed in triplicate. The fluorescence intensity was measured at λ-emission 520 nm upon λ-excitation 380 nm. For Thioflavin T (ThT) assay, 3 μl of protein (0.5 and 0.6 μg/μl, respectively) and 247 μl of 20 μM ThT prepared in 50 mM glycine-NaOH buffer (pH 8.5) were added in triplicates to the wells. Fluorescence intensity was read at λ-emission 490 nm following excitation at 440 nm using a POLARstar OMEGA plate reader (BMG Lab technologies). Each condition for this assay was performed in triplicate.

Thermal shift assay measurements were performed on a QuantStudio 6 Flex Real-Time PCR instrument (Applied Biosystems) in 96-well plates (Axygen) sealed with transparent adhesive film (BioRad). pfGRP78-NBD and hGRP78-NBD were diluted to a final concentration of 5 µM in DSF assay buffer (25 mM HEPES pH 7.5, 250 mM NaCl, 1 mM β-mercaptoethanol) and nucleoside analogues were added at a final concentration of 0.5 mM (5% DMSO final concentration). Protein-compound samples were aliquoted in four replicates followed by the addition of the fluorescent probe bis-ANS (Invitrogen) diluted to 50 µM in a 20 µL total reaction volume. Temperature was continuously increased from 10°C to 95°C at an increment of 1°C/min. Melting curves were analyzed using the software Thermal Shift Assay—Curve Rapid and Automatic Fitting Tool (Lee et al., 2019 (link)). T_m was defined as the temperature corresponding to the maximum value of the first derivative of fluorescence. Error bars shown in Figure 4 represent standard error of the mean and were calculated using GraphPad Prism (version 9.3.1) for macOS (GraphPad Software, San Diego, California, United States, www.graphpad.com/).