Heleos 2

Protein samples at 1 mg/mL (15–40 mM) were injected onto an S200 10/300 Increase SEC column (GE, Marlborough, MA), equilibrated with 25 mM HEPES pH 7.5, 400 mM NaCl. The SEC column was coupled to a static 18-angle light scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX) (Wyatt Technology, Goleta, CA). Data were collected every second at a flow rate of 0.5 mL/min. Data analysis was carried out using ASTRA VI, yielding the molar mass for each sample. The light scattering detectors were normalized and data quality were assessed by testing a BSA standard (Pierce).

SEC-MALLS experiments were run in 20 mM HEPES 7.4, 300 mM NaCl buffer. The injected sample comprised 100 µL of EnvSia156 at 1.8 mg mL⁻¹ in 20 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT. Experiments were conducted on a system comprising a Superdex 200 10/30 GL (GE Healthcare) size exclusion chromatography column, a Wyatt HELEOS-II multi-angle light scattering detector and a Wyatt rEX refractive index detector linked to a Shimadzu HPLC system (SPD-20A UV detector, LC20-AD isocratic pump system, DGU-20A3 degasser, and SIL-20A autosampler). Work was conducted at room temperature (20 ± 2 °C). All solvents and buffers were 0.2 µm filtered before use and a further 0.1 µm filter was present in the flow path. Shimadzu LC Solutions software was used to control the HPLC and Astra V software for the HELEOS-II and rEX detectors. All data were analyzed using the Astra V software. Molecular masses were estimated using the Zimm fit method with degree 1. A value of 0.16 mL g⁻¹ was used for protein refractive index increment (dn/dc), after calibration with a 2.5 mg mL⁻¹ sample of BSA.

Molecular masses of LapD protein samples were determined using size exclusion chromatography coupled with static multiangle light scattering (SEC-MALS) (46 (link)). Purified protein at concentrations between 1 and 15 mg/ml (20 to 300 μM) was subjected to gel filtration on a Bio Sep-SEC-s 3000 column (Phenomenex, Torrance, CA) that was equilibrated in MALS buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl). For samples with c-di-GMP in the mobile phase, 50 μM c-di-GMP was added to the MALS buffer. The SEC was coupled to a static 18-angle light-scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX; Wyatt Technology, Goleta, CA). Data were collected at 25°C each second for 30 min at a flow rate of 1 ml/min. Data analysis was carried out using the program ASTRA V. The detectors were normalized using a sample of 5 mg/ml bovine serum albumin (BSA) (monomeric fraction; Sigma-Aldrich, St. Louis, MO).

Purified CoREST ternary complex that has been gel filtrated was concentrated to > 1 mg/ml. The complex was reapplied to a Superose 6 column. The mass of the complex was detected on elution with an 18-angle MALS light scattering detector (Dawn® HELEOS® II) coupled with a differential Refractive Index detector (Optilab® T-rEX) (Wyatt Technology).

The macromolecular characteristics were determined using size exclusion chromatography (SEC) with three detectors, i.e., the multi-angle light scattering (MALS) (HELEOS II, Wyatt Technology, CA, USA), viscometer (VD) (Viscostar II, Wyatt Technology, CA, USA), and differential refractive index (DRI) (RID 10 A Shimadzu, Japan) detectors. The polysaccharides were solubilized in the eluent LiNO3 (0.1 mol/L) at 2 g/L then, filtered through membrane 0.45 μm, and injected (100 μL) with an automatic injector (SIL-20A, Shimadzu, Japan). The separation was made with two columns OHPAC 804HQ and 806HQ columns (Shodex, Japan) in series. The analysis was performed via a data processing Zimm [34 (link)] “order 1”, using angles from 34.8° to 142.8°. The corresponding value of dn/dc was 0.15 mL/g [35 (link)].