Quartz cuvette

CD spectra were collected on a Chirascan V100 spectropolarimeter (Applied photophysics, Surrey, UK) scanning from 190–250 nm with a 1 nm step and 2 nm bandwidth. 400 µL of TNIP1^417-509 protein at a final 10 µM (0.13 mg/mL) concentration in 50 mM sodium phosphate buffer (pH 8.0) scanned using a quartz cuvette with a 1 mm pathlength (Starna Cells, Atascadero, CA, USA). All spectra were blanked against spectra collected from buffer-only samples. Scans done at increasing temperatures were performed using a Precision Peltier (Quantum Northwest, Liberty Lake, WA, USA) temperature controller. Secondary structure characterization studies were performed using protein samples under the same conditions as above with the addition of 10, 20, 30 or 40% (v/v) 2,2,2-trifluoroethanol (TFE) for 1 h prior to scanning. Post CD analysis done using the DichroWeb server using the Contin-LL (Provencher and Glockner Method) using database 7 from [55 (link)] and references therein. Standards for comparison of AHD1-UBAN versus random coil and pre-molten globule proteins were taken from [45 (link)].

CD spectroscopy is generally used to study chirality of molecules, especially, protein folding and interaction in aqueous media due to the facile manipulation of the experimental conditions and its high sensitivity towards minute variations in temperature, pH and amphiphiles. The protein’s native CD spectra and the changes upon drug titration were obtained using a Jasco 715 spectro-polarimeter (Mary’s Court Easton, MD, USA) and using a quartz cuvette (Starna Cells, Atascadero, CA) with 2 mm path length. All spectra were acquired from 200 to 260 nm and are indicate as the average of three accumulations with the scanning resolution of 0.5 nm. A 50 μL of PDK2 protein (50 μg at 1.5 mg/mL) was transferred to the cuvette with 500 μL of PBS (1×) and 1 μL of monochloroacetic acid (25 mM in DI water) was titrated to the cuvette at each addition. The drug was mixed properly and was incubated three minutes before data acquisition. The titration was terminated when no noticeable change was observed in the spectra pattern. The measured curves were smoothed using Savitzky- Golay algorithm by taking 10 points fitting a second order polynomial curve.

All protein samples were dialysed in 1X Phosphate-buffered saline (PBS) buffer overnight before Circular Dichroism (CD) spectroscopy analysis. Typically, protein samples at 0.01 mg/ml in a quartz cuvette (Starna Cells) of 1.0 cm path length was placed into AVIV 410 spectrometer for CD spectrum acquisition at room temperature (25 °C). Each CD spectrum consisting of the absorbance values was acquired over a wavelength range of 195–260 nm. Each protein sample was measured in three independent scans and the final spectrum is the average of the three scans. To remove absorption background (buffer), three independent scans were made on the 1X PBS buffer. To remove signals from the fused Maltose binding protein (MBP), spectra of independently purified MBP in the same buffer were also acquired that were then subtracted from those of the MBP-Bfr2 fragments. All CD data were expressed as mean residue molar ellipticity in deg × cm2/dmol. The secondary structure content of the proteins was estimated by the deconvolution of far-UV CD spectra according to BeStSel^{31 (link)}. PDB2CD^{32 (link)} was used to convert cryoEM models to CD spectrum data.

CD spectra were measured in a quartz cuvette with an optical path length of 0.5 mm (Starna Cells) using a J-815 spectropolarimeter (Jasco, Japan) at room temperature. The far- and near-UV CD spectra were used to identify changes in protein secondary and tertiary structures. The spectral regions were 200–300 nm. The final spectra were obtained as an average of five accumulations. The spectra were corrected for the baseline by subtracting the spectra of the corresponding polypeptide-free solution. Analogs or IGF-II was dissolved and measured in 5% aqueous acetic acid (0.33 mg/ml; 45 μm).