Ultraflex s

Since purified C-His-tagged TPR domain (Asp24–Arg784) was sequentially degraded during purification and crystallization, we tried to find the stable parts of BcsC-TPR domain using the limited proteolysis analysis. The purified protein was mixed with trypsin on ice at a molar ratio of 100–100,000 to 1. After 0, 30, or 120 min, the mixed samples were checked using SDS-PAGE and analyzed using CBB staining (Fig. S3). The two strongest bands that appeared, were analyzed by N-terminal amino acid sequencing (at the global facility center at Hokkaido University) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Prior to MALDI-TOF-MS, the purified protein was diluted with acetonitrile. The final concentrations of protein and acetonitrile were 25 pmol mL⁻¹ and 50%, respectively. After dilution, 0.65 µL protein sample and 1.3 µL matrix buffer (0.1% triflouroacetic acid and 10 mg mL⁻¹ sinapinic acid in 50% acetonitrile) were loaded on the gold plate. Molecular weight was measured by Ultraflex-S (Bruker Daltonics).

¹H NMR (400 MHz) and ¹³C NMR (100 MHz) were measured in CDCl₃ using a JNM-ESC400 instrument (JEOL Ltd., Akishima, Japan) at ambient temperature. SEC was performed at 40 °C in THF (flow rate, 1.0 mL/min), using a Shodex GPC-101 (Showa Denko K.K., Tokyo, Japan) gel permeation chromatography system (Shodex DU-2130 dual pump, Shodex RI-71 reflective index detector, and Shodex ERC-3125SN degasser) equipped with a Shodex KF-G guard column (4.6 mm × 10 mm; pore size, 8 μm) and two Shodex KF-804L columns (8 mm × 300 mm) in series. TEM measurements were performed with a JEM-2010 (JEOL Ltd., Akishima, Japan) operated at 200 kV. The size distribution of AgNPs was determined by the average of 150–200 observed particles. UV–Vis absorption spectra were recorded on a JASCO 4100 spectrophotometer (JASCO Co., Tokyo, Japan) at 25 °C. Relative UV–Vis absorption intensity (A/A₀ × 100%) of AgNPs without and with NaCl was compared at λ_max (409, 405, and 420 nm for HO–PEG_5k–OH, c-PEG_5k, and MeO–PEG_5k–Ome, respectively). MALDI–TOF mass spectrometry was carried out using a Bruker Daltonics Ultraflex-S at the Open Facility, Hokkaido University, Hokkaido, Japan.