Purification of Cys.
The isolation and purification of Cys from
Rhizobium trifolii TA-1
were carried out as previously described.
Culture supernatants were concentrated fivefold by rotary evaporation, and the concentrated sample was precipitated by adding 3 volumes of ethanol. After centrifugation, the supernatant was concentrated by rotary evaporation and the product was collected by adding 7 volumes of ethanol. After decanting the supernatant, the precipitates were applied to Bio-Gel P-6 column. The fractions were assayed for carbohydrates using the phenol-sulfuric acid method. The fractions that contained Cys were pooled, concentrated, and desalted using Bio-Gel P-4 column. The desalted Cys were confirmed by NMR spectroscopy and MALDI-TOF MS.
Synthesis of QA–Cys.
Cys (1 g) was dissolved in 7% NaOH aqueous solution for 30 min, and 2,3-epoxypropyltrimethylammonium chloride (3.1 g) was added. The mixture was reacted for 5 h at 65 °C, neutralized with HCl and evaporated to a viscous residue. After desalting on a Bio-gel P-2 column, the product was lyophilized, and the final structure was elucidated with EA (ThermoFinnigan, Flash2000) and NMR spectroscopy.
Synthesis of CM–Cys.
CM–Cys was prepared as described in a previous report.
Cys (500 mg) and NaOH (2.8 g) were mixed in distilled water (7.4 mL). Monochloroacetic acid solution (20%) was added. After reacting for 4 h at 50 °C, the mixture was neutralized with 6 M HCl. The mixture was precipitated with 5 vol MeOH and left overnight at 4 °C. The precipitate was dissolved in water and desalted on a Sephadex G-10 column. The product was confirmed using MALDI-TOF MS (Voyager-DETM STR Biospectrometry Workstation) and NMR spectroscopy.
Nuclear Magnetic Resonance (NMR) Spectroscopy.
For the NMR spectroscopic analysis, a Bruker Avance 500 spectrometer was used to record the
C-NMR, DEPT and HSQC spectra. The HSQC spectrum was measured with a spectral width of 3401 Hz in both dimensions and 256/2048 complex data points in
, respectively. NMR analyses were performed in D
O at room temperature.
Scanning Electron Microscopy (SEM).
QA–Cys (3 mM) and CM–Cys (3 mM) were mixed in 100 μL of 20 mM phosphate buffer (pH 7). Hexane was added to a QA–Cys/ CM–Cys mixture in phosphate buffer (pH 7), and a turbid solution was obtained by vortexing for 5 min. After centrifugation, the precipitate was lyophilized. The lyophilized samples were mounted onto stubs using double-sided adhesive tape and then made electrically conductive by coating with a thin layer of gold. The surface morphologies of the materials were examined under a scanning electron microscope (Jeol, JSM 6380, Tokyo, Japan).
Transmission Electron Microscopy (TEM).
QA–Cys (3 mM) and DCA (3 mM; Sigma Aldrich) were mixed in 800 μL of 20 mM phosphate buffer (pH 7). After sonication for 30 s, the aqueous suspension (10 μL) containing the supramolecular aggregates formed by the QA–Cys/DCA mixture was adsorbed onto a carbon-coated copper grid (300-mesh) and air-dried for 1 min. For clear negative staining, the supernatant of 2% uranyl acetate following centrifugation at 13,200 rpm for 2 min was used. The aggregates were examined using a transmission electron microscope (JEOL, JEM 1010, Tokyo, Japan).
Dynamic Light Scattering (DLS).
DLS measurements were carried out with a Wyatt Technology DynaPro Plate Reader at constant room temperature.
This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A1A2012568 and NRF- 2011-619-E0002) and supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0006686). SDG.
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