Chirascan plus spectropolarimeter

Circular dichroism (CD) spectroscopy was used to study the folding and the secondary structure content of the sample proteins in solution. CD spectra were measured in H₂O at 0.25 mg/ml before and after the neutron scattering experiments using a Chirascan Plus spectropolarimeter (Applied Photophysics, United Kingdom) and a 0.5-mm quartz cuvette. Melting curves were measured from 293 to 363 K with a heating rate of 1 K/min at 0.25 mg/ml, in a buffer containing 0.8 mM HEPES (pH 7.5), 6 mM NaCl, and 0.4% glycerol.

Room-temperature absorption spectra were measured with a Varian Cary 4000 UV-Vis-spectrophotometer. The Circular-dichroism (CD) spectra were recorded using a Chirascan-Plus spectropolarimeter (Applied Photophysics) at 20 °C. The OD of the samples was 0.8–1/cm at the maximum of the Qy band. All measurements were performed in the same buffers used for the sucrose gradients.

Far-UV (190-280) CD spectra were acquired on a Chirascan Plus spectropolarimeter (Applied Photophysics Ltd) flushed continuously with pure evaporated nitrogen throughout the experiments. Measurements were recorded in a 0.5 mm strain-free rectangular cell using a 2 nm spectral bandwidth, 1 nm step-size and 1 s measurement time-per-point. The concentrations of the protein samples used ranged between 0.1 mg/mL - 0.4 mg/mL and buffer used was 10 mM HEPES and 150 mM NaCl at pH 7.4. All spectra were acquired at 25 °C and buffer baseline corrected before analysis.

The CD measurements of AcrR and palmitate‐added AcrR were conducted in a Chirascan‐plus spectropolarimeter (Applied Photophysics, Leatherhead, UK) at 20 °C using a 1 mm light path cell. All experiments were performed in buffer (20 mm Tris, pH 7.5, and 150 mm NaCl) at a protein concentration of 25 μm. Palmitate titration was conducted five times to measure the CD spectra, and the palmitate concentration varied from 0 to 20 μm (a maximum of 80% of the protein concentration). CD scans were taken from 260 to 190 nm with a 1 nm bandwidth and a scan speed of 100 nm·min⁻¹. Three scans were averaged, and the solvent signal was subtracted.

Enzyme reactions for CD spectroscopy were conducted for 5 h at 30 °C in water with 0.4 mm 8-oxogeranial and 1 mm NADPH as substrates and 0.5 μm enzyme. Enzyme was diluted at least 150-fold from a buffered solution. Products were extracted with ethyl acetate. The extract from a 1.6-ml reaction was evaporated and taken up in 200 μl of hexane. Completeness of the reaction was verified by GC-MS. Spectra were recorded in 1-nm steps with 0.5-s averaging time on a Chirascan Plus spectropolarimeter (Applied Photophysics) at 20 °C in a 1-mm cuvette. Three measurements were averaged, and background with only hexane was subtracted.

CD spectra were measured in the range from 320 to 190 nm with a Chirascan-plus spectropolarimeter (Applied Photophysics, Leatherhead, UK). The instrument was calibrated with 10-camphorsulfonic acid ammonium salt, and a ratio of 2:1 was found between the positive CD band at 290 nm and the negative CD band at 192 nm. The measurements were carried out in bidistilled water at 20 °C using 0.1- and 1-cm path length cells in amide and aromatic regions, respectively. In the amide region (190–240 nm) of the CD spectrum, molar ellipticity [θ]_λ (deg cm² dmol⁻¹) was calculated as [θ]_λ = θ_obs × MRW/10 × C × L, where θ_obs is the observed ellipticity in degrees, MRW is the mean amino acid residue weight of 110 Da, L is the path length (cm), and C is the protein concentration (g/mL). In the aromatic region (240–320 nm) of the CD spectrum, results were expressed as molar ellipticity (the average molecular masses of RpII, RTX-III, and RTX-VI are 5378, 5287, and 5240 Da, respectively). The secondary structure contents were determined according to the method of Provencher and Glöckner, using their CONTIN program [49 (link)].

The secondary structure of Py4 was studied by CD spectroscopy analysis on a Chirascan Plus spectropolarimeter v.4.4.0 (Applied Photophysics Ltd., Leatherhead, Surrey, UK). The spectra were measured at room temperature from 185–260 nm by using a quartz cell of 1.0 mm thickness and the spectra from 190–260 nm were analyzed. Data were collected at 1 nm intervals with a scan rate of 2 nm/s and expressed as delta epsilon (cm⁻¹M⁻¹) calculated as [θ × (MRW × 0.1)/(C × L)/3298], where θ is the ellipticity (in millidegrees), C is the concentration (in mg/mL), L is the pathlength (in cm), and MRW is the mean residue weight (in Da).