Sypro orange

For thermal denaturation, samples containing 2.0 μM protein and 5× SYPRO Orange (diluted from a 5000 × stock supplied in DMSO; catalogue number S5692, Sigma–Aldrich) were prepared in sample buffer [50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP and 5× SYPRO Orange (from Sigma–Aldrich^®)]. Denaturation curves were monitored in 96-well PCR plates in a Roche LightCycler 480 II, using 465 and 580 nm filters for excitation and emission wavelengths, respectively. Temperature midpoints (T_m) for each folded to unfolded transition were determined by non-linear regression fitting of a modified Boltzmann model (27 (link)) to normalized data in Prism5 (GraphPad Software).
where: a_n and a_d are the slopes, b_n and b_d the y-intercepts, of the native and denatured baselines, respectively. T_m is the melting temperature and m a slope factor.
For circular dichroism, spectra were measured at 20°C between the wavelengths 198 and 280 nm in a JASCO J-715 spectropolarimeter attached to a JASCO PTC-384W temperature control system. CD spectra were measured using a 0.1 mm path length cell (Starna Scientific), with protein at a concentration of 54 μM, that had been buffer-exchanged into 10 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP. Spectra were measured using a 0.1 mm path length cell (Starna Scientific) and represent the average of 10 consecutive scans, where the signal from buffer alone has been subtracted.

Thermal shift assays were performed to determine the melting temperatures of recombinant HpHtrA proteins (wt, S₁₆₄A, D₁₆₅A, S₁₆₆A, D₁₆₈A) in the presence of increasing concentrations of ZnCl₂ or CuCl₂. A change in the melting temperature in the presence ZnCl₂ or CuCl₂ indicates stabilization or destabilization of the protein and is therefore evidence for binding of Zn⁺⁺ or Cu⁺⁺ to HpHtrA. Recombinant HpHtrA proteins (wt, S₁₆₄A, D₁₆₅A, S₁₆₆A, D₁₆₈A) in 50 mM HEPES buffer (pH 7.4) were mixed with SYPRO Orange (Sigma-Aldrich, Vienna, Austria) to final concentrations of 4 µM HpHtrA and 7 × SYPRO Orange in a 96-well PCR plate. Where indicated, increasing concentrations of ZnCl₂ and CuCl₂ were added. Fluorescence data was collected using an Applied Bioscience StepOne Plus PCR cycler (Thermo Fisher Scientific, Schwerte, Germany) during a temperature ramp from 25–95 °C (0.5 °C per min) with a 4 min hold at 25 °C and 95 °C. SYPRO Orange binds to exposed hydrophobic regions within the denatured HpHtrA proteins, which increases the fluorescence of the dye^{57 (link),58 (link)}. The fluorescence emission signal can then be used to determine the melting temperature of the protein, which is located at the infliction point of the fluorescence signal curve. The experiment was performed in triplicates.

In order to improve the stability of the purified AsFur, thermal stability in various buffer systems and salt concentrations was investigated by a Thermofluor assay (Ericsson et al. 2006 (link)). Protein unfolding and its melting temperature (T_m) is monitored by using the fluoroprobe SYPRO Orange dye which emits fluorescence upon binding to exposed hydrophobic regions.
The buffer screen contained 24 buffers covering a pH range from 4.5 to 9.0. Briefly, 5 µl protein (2.5 mg/ml), 12.5 µl 2 × buffer solution (100 mM) and 7.5 µl 300 × SYPRO® Orange (Sigma Aldrich) were mixed and added to the wells of a 48-well PCR-plate (Bio-Rad). To assess the effect of various salts, 15 µl of protein (0.8 mg/ml) diluted in the appropriate buffer (Tris pH 7.5) were mixed with 7.5 µl of 300 × SYPRO® Orange (Sigma Aldrich) and 2.5 µl different salts in concentrations ranging from 0.1–2.0 M. The plates were sealed with Microseal® 'B' Adhesive Seals (Bio-Rad) and heated in a MiniOpticon Real-Time PCR System from 20 to 80 °C in increments of 1 °C per sec. Melting curves were monitored with a charge-coupled device (CCD) camera with wavelengths for excitation and emission at 490 and 575 nm, respectively. T_m, corresponding to the midpoint of the transition curve, was determined using the supplied instrument software and monitoring the fluorescence of the HEX channel.

Deuterium oxide (99.96%) was from Euriso-Top (Saint-Aubin Cedex, France). U, UMP, uracil, K₂HPO₄, KH₂PO₄, NaHCO₃, MnCl₂, HEPES, NaCl, IPTG, imidazole, tetra-n-butylammonium bromide, glycerol, and ATP were from Carl Roth (Karlsruhe, Germany). Glc1,6diP and SYPRO orange, was from Sigma Aldrich (St. Louis, USA). Rib5P was purchased from Biosynth (Staad, Switzerland). Expression vectors (pet15b or pet28a+) containing the genes for YeiN, DeoB, UP, and Yjjg were from Genescript (Leiden, The Netherlands).

Thermal shift assays were performed using a Mini Opticon Real Time PCR machine (BioRad). 0.6 mg/mL protein in PBS was mixed with SYPRO Orange (Sigma-Aldrich, Merck, #S5692) and glycan ligand (10 mM GalNAc; Carbosynth, #MA04390; 10 mM GlcNAc, Carbosynth, #MA00834; 10 mM blood group H type-2 tetrasaccharide; Elicityl, GLY032-2) in a total reaction volume of 25 µL. The temperature was raised by 1 °C/min from 25 to 100 °C, and fluorescence readings were taken at each step.