Quartz suprasil cell

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 spectra were recorded on a Jasco 810 (Japan) dichrograph in quartz suprasil cells (Hellma) with an optical path of 1 mm for peptide in solution or in the presence of liposomes vesicles. Same concentrations of peptide (40 µM) were used for each condition. Spectra were obtained from 3 accumulations between 190 and 260 nm with a data pitch of 0.5 nm, a bandwidth of 1 nm and a standard sensitivity.

SRCD measurements were carried out using the AU-CD beam line of the ASTRID2 synchrotron light source at Aarhus University, Denmark (Miles et al., 2007 (link), 2008 (link)). INpro-9R and INpro16R samples were prepared in a 20 mM NaPhos pH 7.5, 150 mM NaF buffer and spectra measured using a nominally 0.1 mm path length cell (quartz Suprasil cell, Hellma GmbH & Co., Germany), with the precise length determined using interferometry measurements (Hoffmann et al., 2016 ). The concentration of the samples were determined from the absorbance at 205 nm (Anthis and Clore, 2013 (link)), which is measured simultaneously with the CD spectrum. Spectra were measured at 25°C in triplicate in the wavelength range of 170 to 280 nm in steps of 1 nm and a dwell time of 2 s, with a corresponding baseline measured on the buffer alone. SRCD data were deconvoluted using the CDSSTR analysis programme and SMP180 reference datasets at the DichroWeb portal (Whitmore and Wallace, 2004 (link), 2008 (link)). The secondary structure content for the 7 repeats representing the difference between the INpro-9R and INpro-16R constructs was calculated based on the SRCD data given the cumulative nature of SRCD data.

CD experiments were performed at 25 °C using a Chirascan V100 (Jasco, Easton, MD, USA). Protein or peptide samples at 0.1–10 mg mL⁻¹ were examined in a 1.0 mm‐path length Suprasil (quartz) cell (Hellma UK). CD spectra were collected over a wavelength range of 180–260 nm in 1 nm steps using 1s time per point with a bandwidth of 1 nm. All CD spectra were corrected for the baseline by subtracting the spectra of the corresponding IL‐6R or IL‐21R solutions and representing the average of three runs. The content of the secondary structures was estimated with the CDNN v2.1 software.^[40]

The SRCD spectra for EfeM_Psy–metal titration studies were recorded at 25 °C on Beamline B23 at Diamond Light Source. Purified protein, at a concentration of 10 µM in 20 mM HEPES pH 7.4 was used for recording the spectrum of the titration experiments. For titration studies, the SRCD spectra were measured with a 0.5 mm pathlength, Suprasil quartz cell (Hellma®) in the Far-UV CD region (180–260 nm) with a bandwidth of 1 nm and scan speed of 20 nm/min. A 1–10 µl aliquot of metal stock solution (for each metal such as Cu²⁺, Zn²⁺, Fe³⁺ tested) was added stoichiometrically to the protein solution, until the protein reached saturation, as followed spectroscopically. The raw SRCD spectral data in ellipticity (θ) as milli-degrees collected for Far-UV region (180–260 nm), obtained from either four or eight scans, were further processed and finally expressed in molar circular dichroism (Δε) using average amino acid residue molecular weight of 113 daltons.. The dissociation constant (K_d) and stoichiometry of binding were calculated from the plot of ‘∆A’ Absorbance data points (reported as ∆A = A_L−A_R) at 222 nm against the metal (Zn²⁺, Cu²⁺) concentration. The fitting of SRCD data points was carried out using Hills Equation (Siligardi et al. 2002 (link)) with CDApps software (Hussain et al. 2015 (link)).

Emission spectra of pure TPE-Im were recorded at 25°C on a fluorescence spectrophotometer (FS920, Edinburgh Instruments) equipped with a calibrated photomultiplier in a Peltier (air cooled) housing (R928P, Hamamatsu), with a 450 W continuous xenon arc lamp as the excitation source for steady-state photoluminescence measurements using a quartz cuvette with 1.0 cm excitation path length. Emission spectra of TPE-Im/G4 mixtures were recorded using a ChirascanTM Plus CD spectrophotometer from Applied Photophysics equipped for fluorescence measurements. The measurements were carried out using 4 mm by 10 mm suprasil quartz cells from Hellma Analytics. The spectra were recorded at 20°C between 270 and 700 nm, with a bandwidth of 2.0 nm, time per point 0.5 s. The buffer water solvent was Tris-EDTA at 10 mM Tris-Cl and 1 mM EDTA, pH 7.5, same as CD and UV-vis experiment. All the spectra were treated by using OriginPro 2018 software.