Journal: bioRxiv

Article Title: The role of evolutionarily metastable oligomeric states in the optimization of catalytic activity

doi: 10.1101/2022.09.13.507756

Figure Lengend Snippet: (A) Table showing residues in trimer-interface variants, predicted molecular weight (MW) and oligomeric state determined by size-exclusion chromatography (M=monomer, D=dimer, T=trimer). Proteins were injected onto the SEC column at 8 mg/mL. Associated SEC-MALLS data is provided in ( Supplementary Figure 1 ). Residues found in PaCDT are shaded dark grey. Residues not found in either AncCDT-5 or PaCDT are shaded light-grey. (B) Normalized refractive index chromatograms showing elution peaks of trimer-interface variants. Vertical lines aligned with elution peaks of -AncCDT-5 and PaCDTΔC are shown for reference. (C) Schematic showing mutational pathways linking AncCDT-5 and PaCDT via the interface variants in this study (colored by predicted oligomeric state). (D) From left to right, structures of the monomeric (AncCDT-5, PDB 5T0W), dimeric (Alpha-Fold2 model of A5.1+D101F+P218V) and trimeric (PaCDT, PDB 6BQE) forms of the related proteins.

Article Snippet: Samples collected from IMAC were exchanged into size exclusion chromatography (SEC) buffer (20 mM Na 2 HPO 4 , 150 mM NaCl, pH 7.4) using either a HiPrep 26/10 desalting column (GE Healthcare) or through multiple rounds of concentration and dilution using an Amicon Ultra-15 filter unit with a 10 kDa molecular weight cut-off.

Techniques: Molecular Weight, Size-exclusion Chromatography, Injection, Refractive Index