Structural and Biochemical Insight into a Modular β-1,4-Galactan Synthase in Plants

Small-angle X-ray scattering (SAXS) experiments demonstrated that GalS1 works as a dimer in solution.

SAXS was performed at the SIBYLS beamline at the Advanced Light Source. For SAXS coupled with a multi-angle light scattering in line with size-exclusion chromatography (SEC- SAXS-MALS) experiments, 60 µL containing 10 mg/ml GalS1 in 25 mM Hepes pH 7.5, and 100 mM NaCl was used during the experiments. SEC-SAXS-MALS data were collected at the ALS beamline 12.3.1 LBNL Berkeley, California. The X-ray wavelength was set at λ=1.127 Å, and the sample-to-detector distance was 2100 mm, resulting in scattering vectors, q, ranging from 0.01 Å-1 to 0.4 Å-1. The scattering vector is defined as q = 4πsinθ/λ, where 2θ is the scattering angle. The SAXS flow cell was directly coupled with an online Agilent 1260 Infinity HPLC system using a Shodex KW803 SEC column equilibrated with a running buffer as indicated above with a flow rate of 0.5 mL/min. Each sample was run through the SEC, and three second X-ray exposures were collected continuously during a 30-minute elution. The SAXS frames recorded prior to the protein elution peak were used to subtract all other frames. The subtracted frames were investigated by the radius of gyration (Rg) derived by the Guinier approximation I(q) = I(0) exp(-q2Rg2/3) with the limits qRg<1.5. The elution peak was mapped by comparing the integral of ratios to background and Rg relative to the recorded frame using the program SCÅTTER. Uniform Rg values across an elution peak represent a homogeneous sample. Final merged SAXS profiles, derived by integrating multiple frames at the peak of the elution peak, were used for further analysis, including Guinier plot, which determined aggregation-free state. Eluent was subsequently split 3 to 1 between the SAXS line and a series of detectors, including UV at 280 and 260 nm, multi-angle light scattering (MALS), quasi-elastic light scattering (QELS), and refractometer detector. MALS experiments were performed using an 18-angle DAWN HELEOS II light scattering detector connected in tandem to an Optilab refractive index concentration detector (Wyatt Technology). System normalization and calibration was performed with a BSA monomer using a 45 μL sample at 10 mg/mL in the same SEC running buffer and a dn/dc value of 0.19. The light scattering experiments were used to perform analytical scale chromatographic separations for Mw determination of the principal peaks in the SEC analysis. UV, MALS, and differential refractive index data were analyzed using Wyatt Astra 7 software to monitor the homogeneity of the sample across the elution peak complementary to the above-mentioned SEC-SAXS signal validation.

raw SEC-SAXS frames, final merged SAXS curve, SEC-MALS report structure model of GALs dimer and alternative dimer

2023-01-16

2023-01-18

SEC-SAXS

1.127 Å

2.1 m

Sample:

• Macromolecule 1: β-1,4-Galactan Synthase
• Sample Full Name: GALs
• Sample Type: Protein
• Source Organism: Geobacillus stearothermophilus
• Source Organism NCBI Taxonomy ID:
• Expression System: Escherichia coli
• Expression NCBI Taxonomy ID:
• Sequence or Chemical Formula:
• PNKRIFQAYGNAAALFVQMGAYRGGPTTFAVVGLASKPIHVFRLPWYKCEWISNNGSSIRAKAYKMLPDWGYGRVYTVVVVNCTFPVNPNQDNAGGRLMLNAYYDESQRKYEKFTALEELPGSYNESKFRPPYQYEYLYCGSSLYGNLSASRFREWMAYHAWFFGPSSHFVFHDAGGVSPEVRAALDPWVRAGRATVQDIRGQAEFDGYYYNQFLVVNDCLHRYRYSANWTFYFDVDEYIYLPEGNTLESVLKDFSNYTQFTIEQNPMSSALCFNDSTQDYPRQWGFEKLLFRESRTGIRRDRKYAIQAKNAYATGVHMSENVIGKTLHQTETKIRYYHYHNSIQVPGELCREFLPLSAKNNVTWYNGLPYVYDDNMKKLASTIKDFERNTIG