J 600 spectropolarimeter

UV-vis spectra have been obtained with a Jasco V-530 UV/VIS spectrophotometer. CD spectra have been collected with a JASCO J-600 spectropolarimeter equipped with a thermostatted cell holder. RNA samples in 50 mM Tris-HCl, pH 7.4, 100 mM KCl were 10 μM. The spectra were recorded in 0.5 cm quartz cuvette at increasing temperature. Ordinate is expressed in mdeg.

These spectra were recorded on a Jasco J-600 spectropolarimeter (Jasco, Tokyo, Japan), at 5 °C in quartz cells with 1-cm path length. The spectra were recorded at the bandwidth 1.0 nm and resolution 1.0 nm with a scan speed of 50 nm/min. The scans were accumulated and automatically averaged. The concentration of enzyme in the cell was 1.0 µM. The experiments were carried out in the buffer consisting of 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1.0 mM EDTA and 7% glycerol (v/v). Because of aggregation of F98W hSMUG1 under this condition for this mutant form CD spectrum was recorded in high salt concentration that increases the noise up to 210 nm in the spectrum.

Analytical thin-layer chromatography (TLC) was performed with pre-coated silica gel plates (Silica gel 60 F254, Merck, Darmstadt, Germany), and UV irradiation was used for visualization (254 and 360 nm, Herolab, Wiesloch, Germany).
Semi-preparative reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) was carried out by using a Jasco system (Jasco, Groß-Umstadt, Germany) equipped with a 1-mL sample loop (Rheodyne, CA, USA) at a wavelength of 360 nm with an Agilent Eclipse XDB-C 18 column (250 × 9.4 mm, 5 μm, Agilent, Waldbronn, Germany).
1D and 2D nuclear magnetic resonance (NMR) spectra were recorded at 600 MHz for ¹H NMR and 150 MHz for ¹³C NMR, respectively, using an Agilent DD2 600 MHz spectrometer (Agilent, Waldbronn, Germany). Chemical shifts are reported in parts per million (ppm), coupling constants (J) are given in Hertz (Hz), and CD₃CN was used as the solvent.
High-resolution mass spectrometry with electrospray ionization (ESI-HRMS) data were obtained using an LTQ Orbitrap XL instrument (Thermo Fisher Scientific, Bremen, Germany). UV spectra were obtained with a J-750 Spectrophotometer (Jasco, Groß-Umstadt, Germany) in acetonitrile (MeCN).
For circular dichroism (CD) measurements, a J-600 Spectropolarimeter (Jasco, Groß-Umstadt, Germany) with a 0.1-cm cell in MeCN at room temperature was used.

Circular dichroism (CD) assays were carried out at room temperature (25 °C) in normal saline and in 20% trifluoroethanol using a Jasco J-600 spectropolarimeter. Spectra were recorded in the range a 195–260 nm, using a 1 mm path-length quartz cell. Each spectrum was obtained by averaging three scans with 1 nm step and 2 nm spectral bandwidth. The samples TBI_tag and nTBI have the same optical absorption at 214 nm.
The fractions of the secondary structure elements were calculated by minimizing the difference between the theoretical and experimental curves by varying of the impacts of the α-helixes, β-sheets, turns and non-structured forms. Theoretical values at every wavelength were the linear combination of the basis spectra of every type of secondary structure [48 (link)].

CD spectra were measured on a Jasco J-600 spectropolarimeter, at room temperature. Pathlengths of 1 and 10 mm were used for the peptide and aromatic region, respectively. Each spectrum represents the average of at least 7 scans. Concentrations of the solutions were in the range of 0.05–0.07 and 0.5–2.5 mg/ml for the peptide and aromatic region, respectively. Spectra were measured in MeOH, TFE, and water, pH 7 (0.01 M sodium phosphate buffer). The data are presented as total molar ellipticity [θ].

UV-melting was performed by using Jasco V-750 UV-visible spectrophotometer equipped with a Peltier temperature control system (ETCS-761) (Jasco Europe, Cremella, Italy). The spectra were analyzed with Spectra Manager (Jasco Europe, Cremella, Italy). The oligonucleotides (5 μM) were annealed in 50 mM Na-cacodylate, pH 7.4 and 100 mM KCl, (5 min at 95 °C, overnight at RT). The melting curves were recorded at 295 nm in a 0.5 cm path length quartz cuvette, heating (25–95 °C) at a rate of 0.5 °C/min.
Circular Dichroism (CD) spectra were obtained on a JASCO J-600 spectropolarimeter, equipped with a thermostated cell holder, with 5 μM oligonucleotide solutions in 50 mM Na-cacodylate, pH 7.4, 100 mM KCl. The spectra were recorded in 0.5 cm quartz cuvette at 25 and 95 °C and reported as ellipticity (mdeg) versus wavelength (nm). Each spectrum was smoothed and subtracted to the baseline.