Astra 6.1 software package

The extent of the complex formation by the wild-type and deletion mutant αB-crystallin when incubated with CS at 43 °C was examined by light scattering analysis at different time intervals. Samples with a chaperone-to-substrate ratio of 1:2 were incubated at 43 °C for the desired time before evaluation. The reaction mixtures of either αB-wt or αBΔ54–61 with citrate synthase were passed through a TSK G5000PW_XL size exclusion column (Tosoh Bioscience, King of Prussia, PA, USA) connected to a Shimadzu HPLC system with UV and refractive index detectors and coupled to a static multi-angle laser light scattering (MALS) and quasi-elastic light scattering system (Wyatt Technology Corporation, Santa Barbara, CA, USA). The molar mass, size, and polydispersity were determined using ASTRA 6.1 software package (Wyatt Technology).

The molecular weights of the constructs were determined by SEC-MALS detector. The mass measurements were performed on a Dionex UltiMate3000 HPLC system equipped with a three-angle miniDAWN TREOS static light scattering detector (Wyatt Technology). The sample volumes of 100 μl at a concentration of 40 μM were applied to a Superose 6 10/300 GL column (GE Healthcare) previously equilibrated with 20 mM Hepes, pH 7.5, 300 mM NaCl, and 2 mM TCEP at a flow rate of 0.5 ml/min. The data were analyzed using the ASTRA 6.1 software package (Wyatt Technology), using the refractive index of the buffer as a baseline and the refractive index increment for protein dn/dc = 0.185 ml/g.

The determination of the macromolecular magnitudes (i.e., the mass average molar mass (M_w), number average molar mass (Mn), gyration radius (Rg), hydrodynamic radius (Rh) and intrinsic viscosity ([η]) of EPS in the diluted regime was carried using high pressure size exclusion chromatography (HPSEC) [18 (link)]. The HPSEC-MALLS was equipped with three detectors online: a multi-angle laser light scattering (MALLS) filled with a He–Ne laser at 690 nm and a K5 cell (50 μL) (HELEOSII, Wyatt Technology, Goleta, CA, USA), a differential refractive index (DRI) (RID10 A Shimadzu, Japan) as well as a viscosimeter (Viscostar II, Wyatt Technology, CA, USA). Columns (OHPAK SB-G guard column, OHPAKSB806 and 804 HQ columns (Shodex, Showa Denko, Japan)) were eluted with LiNO₃ (0.1 M) at a flow rate of 0.7 mL/min. Note that the solvent was filtered through a 0.1 μm filter unit (Millipore, Burlington, MA, USA), degassed and filtered using a 0.45 μm filter upstream column. EPS was solubilized at a concentration of 2 g/L in 0.1 M of LiNO₃ solution for 24 h under stirring, filtered (0.45 μm), and then injected through a 500 μL full loop. The collected analyses data were analyzed involving a Astra 6.1 software package (Wyatt technology, CA, USA) and a dn/dc of 0.15 mL/g.