Astra6 program

The molecular masses of CouR and CouR–DNA complexes were determined using SEC-MALS. A total of 50 μl of 30 μM CouR in 20 mM HEPES, pH 7.0 and 50 mM potassium chloride was injected into an HPLC 1260 Infinity LC (Agilent Technologies) equipped with a Superdex 200 10/300 column (GE Healthcare). The column was operated at room temperature and a flow rate of 0.2 ml min⁻¹. Data were collected with the miniDAWN TREOS multiangle static light scattering device and Optilab T-rEX refractive index detector (Wyatt Technologies). Data were analysed using the ASTRA6 program (Wyatt Technologies).

First, SEC experiments were carried out on a Varian ProStar 215 solvent delivery system coupled to a Varian ProStar 320 UV-visible detector. Separation was performed using BioSep 5 μm SEC-s2000 145 Å, 300 × 4.6 mm LC column (Phenomenex, Torrance, CA, USA). 50 mm sodium phosphate buffer, pH 6.65, containing 150 mm NaCl was used as an eluent at a 0.5 ml/min flow rate. met–Evasin-4 was injected at 0.1 mg/ml concentration and followed by absorbance at λ = 280 nm.
In addition, SEC-MALS was used to determine the oligomeric state of Evasin-4 by injecting 100 μl of protein at 5.3 mg/ml onto a Superdex 75 10/300 gel filtration column (GE Healthcare) in 20 mm Tris, 100 mm NaCl, pH 7.3, at 17 °C. SEC was performed with online static light scattering (miniDAWN TREOS, Wyatt Technology) and differential refractive index determination (Shimadzu RID-10A) on a Shimadzu HPLC system. Protein concentration at elution and massed average molecular mass was determined using standard protocols in the ASTRA6 program (Wyatt). The set-up was calibrated with 100 μl of 5 mg/ml monomeric chicken egg white albumin (Sigma–Aldrich). A refractive index increment (dn/dc) value of 0.185 ml/g was used for Evasin-4.