Sypro orange protein stain

8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (D-007) was purchased from Biolog Life Science Institute, Bremen, Germany. 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), Alamar blue cell viability reagent and SYPRO™ Orange protein stain were purchased from ThermoFisher Scientific, Waltham, MA, USA. Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) sodium salt, 0.33% neutral red solution, dimethyl sulfoxide (DMSO) for molecular biology and anti-Rabbit IgG, HRP conjugate, were obtained from Sigma-Aldrich, St. Louis, MO, USA. Rap1A/Rap1B (26B4) rabbit mAb, EPAC1 (DE3) mouse mAb and STAT3/phospho-STAT3 (Tyr 705) antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.

Thermostability curves were recorded in four replicates per sample on a Thermofluor CFX96 thermocycler.
Purified protein samples were diluted to 0.5 mg/ml in 20 mM sodium phosphate, 150 mM NaCl, pH 7, mixed with SYPRO Orange protein stain (Thermo Fischer Scientific). Denaturation cycle was carried out between 10 °C and 95 °C in increments of 0.5 °C for 10 s. Triplicate curves were collected for all samples.

Purified LdtR was tested with different concentrations of Zn²⁺ (0–50 μM) as described previously [9 (link),31 (link)]. LdtR was diluted in 100 mM Tris buffer pH 8.0 and 150 mM NaCl to a final concentration of 30 μM. The reagent SYPRO orange protein stain (ThermoFisher, Waltham, MA, USA) was added to a final concentration of 5X. Volumes of 25 μl of a mix containing ligand, protein, buffers, and SYPRO reagent were added in a 96-well plate (Bio-Rad) and ran in triplicate. The samples were first incubated at 25°C for 5 min and then heated to 80°C at a rate of 1°C per min. By measuring the increase in the fluorescence of the SYPRO orange reagent, the unfolding of LdtR was monitored in a multicolor real-time PCR detection system (iCycler iQ, Bio-Rad). The generated fluorescence intensities were plotted against temperature for each sample and the denaturation curves were analyzed and fitted using the Boltzmann equation in Microcal Origin 2017 software (OriginLab, Northampton, MA, USA). The midpoint of each denaturation curve of LdtR in presence of Zn²⁺ was calculated and compared to the midpoint value of LdtR in presence of buffer as a control.

Solutions of either 2PT-NBD1 or F508del-2PT-NBD1 (10 µM final concentration), nanobodies (30 µM final concentration) and 2.5× or 5× concentrated SYPRO Orange Protein Stain (Molecular Probes) diluted in 20 mM HEPES pH 7.5, 150 mM NaCl, 3 mM MgCl₂, 2 mM ATP and 10% (w/v) glycerol, 10% (w/v) ethylene glycol, were added to the wells of a 96-well PCR plates type BR white (VWR) in a final volume of 25 µl. Plates were sealed with EasySeal sheets (Molecular dimensions) and spun 2 min at 900 × g. SYPRO orange fluorescence was monitored in CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using FRET scan mode from 10 to 80 °C in increments of either 1 or 0.2 °C.