J 815 spectropolarimeter

CD spectra were recorded on a Jasco J-815 spectropolarimeter at 25°C in a step-scan mode using 0.1-cm path length quartz cells from Hellma (Mülheim, Germany). The concentration of unlabeled GpA peptide was 5 µM. Data points were collected at a resolution of 1 nm, an integration time of 1 s, and a bandwidth of 1 nm. Each shown spectrum results from at least three averaged scans from which buffer scans were subtracted. The measured ellipticity θ was converted to molar ellipticity [θ] by: where M is the molar mass, L the path length, and C the peptide concentration.
The GpA TM domain was reconstituted in 10 mM phosphate buffer (pH 7.4) containing A8-35 as well as in several solvents containing SDS, DDM, or pure TFE, which are known to stabilize α-helical structures. Prior to CD measurements, samples were treated in the same way as described above for FRET measurements. Secondary structure contents were estimated with the DICHROWEB software [30] (link), [31] (link) using both soluble and TM proteins as reference datasets (CDSSTR method, reference set 7).

The far-UV CD measurements were performed using a Jasco J-815 spectropolarimeter at 20 °C in a 1 mm path length quartz cuvette from Hellma over the wavelength range 200–260 nm. A bandwidth of 1 nm and a scan speed of 20 nm/min were employed. At least 3 consecutive scans were obtained. The resulting data was smoothed and the contribution of the buffer blank was subtracted. The monitored scans were averaged.

CD spectra were recorded on a Jasco J-815 spectropolarimeter by using Hellma 110-QS cuvettes of 1 mm path length. CD measurements were performed in 50 mM Tris-HCl pH 8, 50 mM NaCl using protein concentrations of 2 μM. 20 scans were averaged and the buffer baseline was subtracted.

Far-UV CD measurements were recorded on a JASCO J-815 spectropolarimeter on Hug1 samples (10 μM) dissolved in H₂O by using Quartz SUPRASIL® cells (Hellma) with a path length of 1mm. Far-UV CD spectra of Hug1 were recorded at 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C. All far-UV CD spectra were recorded from 260–190 nm with a scan speed of 50 nm/min and a time response of 1 s. Equivalent spectra of buffer were recorded and substracted from the spectra of the protein. Molar ellipticities per residue were calculated as follows: , with θ the ellipticity in mdeg, l the cell path length in cm, C the concentration in M and n the number of peptide bonds.

CD spectroscopic experiments were used to study the solution structure of analogues in phosphate buffer (10 mM, pH 7.4) and in 50% 2,2,2-trifluoroethanol (TFE) using a Jasco J-815 spectropolarimeter in Hellma QS cuvettes with pathlength 1.00 mm. Stock solutions of 1 mg/mL were prepared from lyophilized analogs in 50 mM Na₂HPO₄, pH 7.4 and were diluted to appropriate concentrations of ~0.1 mg/mL in 10 mM buffer, or same concentration in 50% TFE (v/v). The spectra of the analogues were recorded in 10 mM buffer and 50% (v/v) TFE at room temperature. The measurements were conducted in the far UV range from 250 to 190 nm with a scan rate of 20 nm/min at 1 nm bandwidth, and a time constant of 0.5 s. The resulting spectra were the average of six separate recordings. Blank samples of 10 mM buffer, respectively 10 mM buffer with 50% (v/v) TFE, were recorded and subtracted from the relevant sample spectra. The CD spectra were normalized with regard to the UV absorbance at 280 nm (relative to the D2D analog). The UV absorbance data were measured on a Shimadzu UV-3600 UV–vis–NIR spectrophotometer using a 10 mm Hellma quartz cuvette. Finally, the CD spectra were smoothed with a five-point Savitzky–Golay filter in Jasco spectra analysis and the CD signal at 250 nm was adjusted to zero.