J 815

CD spectra of around 0.142 mg/ml of nRhi o 1 and rRhi o 1 were recorded in Jasco Corp. J-815 CD spectropolarimeter at 20°C within a wavelength range of 260–200 nm. Average spectra of five scans were analyzed in K2D of Dichroweb server [25 (link)].

The assay followed a procedure described earlier11 (link). In brief, recombinant T. hominis proteins were expressed in E. coli with a His-tag and purified by Ni-NTA affinity chromatography followed by gel filtration (Äkta Purifier System 10, Column 16/60 Superdex 200 pg, GE Healthcare). Samples for the in vitro Fe/S-cluster synthesis assay were prepared in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI, USA). Protein solutions and reagents were incubated under anaerobic conditions over night at 10 °C before the experiments. The 300 μl standard reaction contained 2.5 μM ThNfs1–ThIsd11, 3 μM ThYfh1, 3 μM ThYah1, 0.3 μM human FdxR and 100 μM of either ThIsu1 or CtIsu1 in buffer R (35 mM Tris–HCl, pH 8, 150 mM NaCl, 0.2 mM MgCl₂, 20 μM PLP, 0.5 mM NADPH, 0.5 mM sodium ascorbate, 0.3 mM FeCl₂). The reaction was transferred to a CD cuvette, sealed tightly and placed at 30 °C in a CD spectrophotometer equipped with an automatic stirring device (J-815, Jasco). After 2 min of temperature equilibration the Fe/S-cluster synthesis reaction was initiated by anaerobic addition of 0.5 mM cysteine. The CD signal change at 431 nm was recorded. Subsequently, full spectra were recorded from 300 to 650 nm. Initial rates were estimated by a linear fit to the initial 4.5 min of the reaction. Evaluation of the data were carried out using Origin 8 G software.

Optical rotations were obtained on a JASCOP-1020 digital polarimeter. UV spectra were recorded on Waters 2487, while the ECD spectrum was measured on the JASCO J-815 spectropolarimeter. IR spectra were taken on a Nicolet NEXUS 470 spectrophotometer as KBr disks. ¹H NMR, ¹³C NMR, DEPT, and 2D NMR spectra were recorded on an Agilent 400 MHz DD2 spectrometer. HRESIMS data were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. MPLC was performed using a C₁₈ column (Agela Technologies, YMC-Pack ODS-A, 3 cm × 40 cm, 5 μm) at a flow rate of 20 mL/min. Preparative HPLC collection used a C₁₈ column (Waters, YMC-Pack ODS-A, 10 mm × 250 mm, 5 μm, 3 mL/min). Aniline was purchased from Aladdin (Shanghai, China).

Circular dichroism (CD) was used to determine peptide folding in solutions containing 0–90% (vol%) of trifluoroethanol (TFE) to mimic surface interactions. Spectra were recorded with a CD spectrometer (JASCO, J-815) at room temperature, using a 1.0 mm cuvette. Each peptide sample was dissolved at 0.2 mg/mL in 10 mM potassium phosphate containing 100 mM (NH₄)₂SO₄ (pH 7.4) with and without TFE at 4 °C for 16 h. A spectral range was acquired from 185 to 300 nm at a scanning speed of 60 nm/min for each sample and reported values represent an averaged spectrum across triplicate measurements. The secondary structure Far-UV CD spectra were processed with the tools of CD Pro [71 (link)]. The mean residue ellipticity of each sample was converted to mean residue absorbance and further processed with CRDATA.exe to create the input file for SELCON3.exe, CDSSTR.exe and CONTILL.exe. For each set of data, the reference set selected was SMP50. The fractions of secondary structure (Regular Helix, Distorted Helix, Regular Sheet, Distorted Sheet, Turns and Unordered) were averaged for all three CD Pro tools (SELCON3, CDSSTR, and CONTILL).

Far‐UV CD spectra (190–260 nm) were recorded on Jasco J‐815 CD equipped with a Jasco CDF‐4265/15 Peltier unit with a cell holder thermostated by circulating water from a Neslab RTE‐111 water bath. Spectral measurements were carried out using Ct‐frataxin protein concentration 22 μm in different buffer conditions using QS quartz 1 mm demountable cuvettes (Hellma analytics, Müllheim, Germany). Baseline correction was obtained by subtraction of the appropriate buffer spectrum. Thermal unfolding curves were obtained by monitoring the ellipticity at 222 nm with a heating rate of 2 °C·min⁻¹ in the temperature range 25–95 °C. The apparent T_m was calculated assuming a two‐state mechanism of unfolding and using nonlinear regression analysis as described by in 31.