Chirascan cd spectropolarimeter

Far- and near-UV CD spectra of HSP18 at different temperatures between 25–43°C were recorded using Chirascan CD spectropolarimeter (Applied Photophysics, Leatherhead, UK) equipped with peltier system as described previously [29 (link)]. Protein concentrations used in far- and near-UV CD experiments were 0.2 mg/ml (in 10 mM phosphate buffer, pH 7.5) and 0.5 mg/ml (in 50 mM phosphate buffer, pH 7.5), respectively. Far-UV CD spectra were collected from 195–260 nm using a quartz cell with 1 mm path length, while the near-UV CD spectra were collected from 250–300 nm using 10 mm path length cell. Scan rate of 50 nm/min, data pitch of 0.5 nm, band width of 1 nm and response time of 0.2 seconds were used for far- and near- UV CD data collection. The reported spectra for far- and near-UV CD are the mean of five scans. For both experiments, spectra were recorded after incubating HSP18 at respective temperature for 1 hr. The curve-fitting program CONTINLL was used to analyze the secondary structural content of HSP18 at different temperatures [29 (link)].

All the reagents involved in this research were commercially available and used without further purification unless otherwise noted. Solvents were either employed as purchased or dried before use by standard laboratory procedures. Thin-layer chromatography (TLC) was carried out on 0.25 mm Leyan silica gel plates (60F-254). Column chromatography was performed on silica gel (200-300 mesh) as the stationary phase. ¹H, ¹³C NMR, 2D NMR spectra were performed on Bruker Avance-500 NMR spectrometers. Chemical shifts are reported in ppm with residual solvents or tetramethylsilane (TMS) as the internal standards. The following abbreviations were used for signal multiplicities: s, singlet; d, doublet; dd, doublet of doublet; m, multiplet. Host-guest complexes were prepared by simply mixing the guests and hosts in 1: 1 stoichiometry in the corresponding solvent. Electrospray-ionization high-resolution mass spectrometry (ESI-HRMS) experiments were conducted on an applied Q-EXACTIVE mass spectrometry system. Circular Dichroism (CD) and UV-Vis spectra were recorded on an Applied Photo Physics Chirascan CD spectropolarimeter, using a 1 cm quartz cuvette. Fluorescent spectra were recorded on a spectrofluorometer (Edinburgh FS5), using a 1 cm quartz cuvette. Specific rotations were measured on Rudolph Research Analytical Autopol I Polarimeter (589 nm) in a 1 dm length cell under 25 °C.

200 μL of 10 μM oligonucleotide were prepared in assay buffer and annealed as described above. CD spectra were recorded on an Applied Photo-physics Chirascan CD spectropolarimeter using a 1 mm path length quartz cuvette. CD measurements were performed at 298 K over a range of 200–320 nm using a response time of 0.5 s, 1 nm pitch and 0.5 nm bandwidth. The recorded spectra represent a smoothed average of three scans, zero-corrected at 320 nm (Molar ellipticity θ is quoted in 10⁵ deg cm² dmol⁻¹). The absorbance of the buffer was subtracted from the recorded spectra.

CD spectra of the PSP and PSP−Zn (2.0 mg/mL) were obtained on a Chirascan CD spectropolarimeter (Applied Photophysics Ltd., Surrey, UK) over the wavelength range of 190–260 nm at a scan rate of 100 nm/min and a bandwidth of 2.0 mm.