Dawn 8

For size exclusion chromatography combined with multiangle light scattering (SEC-MALS) analysis of DBB and DBB-ANK domains, 50-μl protein samples at a concentration of 2 mg/ml were injected onto a Superdex 200 Increase 10/300 GL column (GE Healthcare). Differential refractometry and multiangle light scattering data were collected using Optilab T-rEX (Wyatt technology) and DAWN8⁺ (Wyatt technology) instruments, respectively. Data analysis was performed using ASTRA (v6.1) software (Wyatt technology).

SEC-MALS experiments were performed at room temperature. For each experiment, 100 μl of protein at 3 mg/ml was injected onto a Superdex 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated with 20 mm HEPES, 150 mm NaCl, and 2 mm DTT at a flow rate of 0.5 ml/min. The static light scattering, differential refractive index, and the UV absorbance at 280 nm were measured in-line by DAWN 8+ (Wyatt Technology), Optilab T-rEX (Wyatt Technology), and Agilent 1260 UV (Agilent Technologies) detectors. The corresponding molar mass from each elution peak was calculated using ASTRA 6 software (Wyatt Technology).

¹⁵N‐labeled MOZART1 protein buffered in 50 mM sodium phosphate pH 6.0, 150 mM NaCl was analyzed on a Superdex 200 Increase 5/150 GL (GE Healthcare) column with multiangle light scattering (MALS). The column was equilibrated in a 50 mM sodium phosphate 0.1 μm filtered buffer (pH 6.0 and 150 mM NaCl) on an Agilent 1260 Infinity LC chromatographic system (Agilent Technology). Data were collected using a DAWN 8+ and Optilab T‐rEX refractive index detector (Wyatt Technology). Of 150 µM protein sample, 70 µL was loaded on the column and the separation was performed at a flow rate of 0.4 mL/min at 15°C. Results were analyzed using the ASTRA 6.1 software (Wyatt Technology).

Number average molecular weight ( ), weight-average molecular weight ( ), and polydispersity of polymer precursors and polymer conjugates were measured using size-exclusion chromatography (SEC) on a HPLC Shimadzu system equipped with UV, an Optilab® rEX differential refractometer and multi-angle light scattering DAWN® 8™ (Wyatt Technology, USA) detectors. For these experiments, 0.3 sodium acetate buffer pH = 6.5 and Superose 6 column were used. The content of thiazolidine-2-thione (TT) groups was determined spectrophotometrically on a Specord 205 (Jena Analytics) spectrophotometer ( ; methanol). The content of pyridyldisulfide groups (PySS) in the polymer conjugates was determined by UV spectrophotometry after reaction with dithiothreitol [52] (link). The content of maleimidyl groups (mal) in polymer conjugates was determined by a modified Ellman€s assay, as a difference between concentration of cysteine in solution before and after reaction with maleimidyl groups of the polymer [53] (link). The characteristics of polymer conjugates are summarized in table 2.

Ifit proteins (1 mg/ml) were analyzed by SEC on a Superdex 200 Increase 10/300 GL column at 4 ˚C at a flow rate of 0.5 ml/min. Ifit complexes were examined by combining Ifit proteins at stoichiometric concentrations for 1 h at 4 or 30 ˚C, before SEC analysis. 280-nm absorbance was normalized such that peak height was equal to 1, for ease of comparison. For SEC-MALS, Ifit1b (0.5 or 2 mg/ml in a 150-μl loop) was applied to a Superdex 200 Increase 10/300 GL column at room temperature, at a flow rate of 0.4 ml/min. MALS analysis was performed by inline measurement of static light scattering (DAWN 8+; Wyatt Technology), differential refractive index (Optilan T-rEX; Wyatt Technology), and 280-nm absorbance (Aligent 1260 UV; Aligent Technologies). Molecular mass was calculated using the AS-TRA6 software package (Wyatt Technology). Access to SEC-MALS apparatus was kindly provided by Dr. Janet Deane.

For the Size-Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS) analysis, the purified PorX_Fj (1.6 mg mL⁻¹) was incubated for 1 h at RT with 20 mM acetyl phosphate (AcP) + 10 mM MgCl₂, or 20 mM AcP, or 10 mM MgCl₂, or 100 µM ZnCl₂. The samples were loaded on a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS at a flow rate of 0.6 mL min⁻¹, using an Ultimate 3000 HPLC system (Fischer Scientific). Detection was performed using an eight-angle light-scattering detector (DAWN8, Wyatt Technology) and a differential refractometer (Optilab, Wyatt Technology).
For the Size-Exclusion Chromatography (SEC) analysis, the purified PorX_Fj (1.6 mg mL⁻¹) was incubated for 1 h at RT with 100 µM ZnCl₂, MnCl₂, or CuCl₂. The samples were loaded on a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS supplemented with100 µM of the corresponding metal solution.