J 815 spectrometer

CD spectra were collected using a Jasco J-815 spectrometer (Jasco). Protein solutions were buffered with 10 mm sodium phosphate, pH 7.2. Protein concentrations were calculated by measuring the absorbance at 280 nm in conjunction with the predicted extinction coefficient of 30,940 m⁻¹cm⁻¹ for both CBM and CBM-CT. The extinction coefficient was calculated using Protparam from the ExPASY server (31 ), which employs an Edelhoch based method (32 (link)– (link)34 (link)). Spectra were acquired using a quartz cuvette with a 0.1-cm path length. The spectra were collected between 260–180 nm at 50 nm min⁻¹ at 0.5-nm increments with a response time of 0.5 s and a data pitch of 0.5 nm. Base lines were collected in the same manner using 10 mm sodium phosphate buffer, pH 7.2. Spectra were averaged over 4 scans then corrected for solvent background and smoothed using a Savitzky-Golay method (35 ). To determine secondary structure components, various deconvolution methods were applied to the data, which were accessed using the DICHROWEB server (36 (link)).

The UV and ECD spectra were recorded on a JASCO J-815 spectrometer (Loveland, CO, United States) in MeOH or MeCN using a 1-cm cell. Scan speed was set at 200 nm/min in continuous mode between 600 and 190 nm. NMR spectroscopic data were recorded on a Bruker Avance Neo 600 MHz NMR spectrometer equipped with a QCI 5-mm cryoprobe and a SampleJet automated sample changer (Bruker BioSpin, Rheinstetten, Germany). One-dimensional (1D) and 2D NMR experiments (¹H, COSY, ROESY, HSQC, and HMBC) were recorded in DMSO-d₆ or CD₃OD. Chemical shifts were reported in parts per million (δ) and coupling constants (J) in Hz. The residual DMSO-d₆ signal (δ_H 2.5; δ_C 39.5) and CD₃OD signal (δ_H 3.31; δ_C 49) were used as internal standards for ¹H and ¹³C NMR, respectively. Trans-caffeic acid (reference C0625-25 g, >98% purity) was purchased from Sigma-Aldrich (Steinheim, Germany), and trans-ferulic acid (reference 9936, 99% purity) was purchased from Roth (Karlsruhe, Germany).

Circular dichroism (CD), X-ray diffraction (XRD), small-angle X-ray scattering (SAXS), and Fourier transform infrared (FTIR) were employed to measure the polypeptide secondary conformation and self-assembled structures. CD measurements of polypeptide samples were conducted at the concentration of 0.1 mg mL⁻¹ on a JASCO J-815 spectrometer (JASCO Corporation, Japan) in a 0.1 mm quartz cell from 190 nm to 260 nm of the wavelength. A Rigaku Ultima IV-9407F701 X-ray spectrometer (Rigaku Corporation, Japan) was used to record XRD patterns of star-shaped polypeptide hydrogel samples. XRD system scanned the patterns from 2θ = 5° to 40° at a speed of 10° min⁻¹ by using radiation (50 kV, 250 mA) and Cu K alpha (0.154 nm). At 25 °C, SAXS profiles of the sol and gel samples were obtained under 4 × 10⁻¹ torr and the voltage and current were controlled in 45 kV and 650 μA, respectively. The SAXS measurements were run on a Bruker diffractometer (NanoSTAR-SHAPED U system, Bruker AXS GmbH, Germany). The samples were contained in 1 mm quartz capillary tubes at different sol and gel concentrations. Before the measurements, a silver behenate was used as a standard sample for calibrating the SAXS. Attenuated total reflectance FTIR spectra of freeze-dried gel samples were recorded on a Thermo Nicolet Nexus 670 FTIR spectrometer (Thermo Electron Corporation, USA).