1260 infinity 2

To assess changes in the oligomeric state of TsaC, in-line SEC–MALS and DLS experiments were conducted. SEC–MALS experiments were conducted using an Agilent 1260 Infinity II HPLC equipped with a 4 °C chilled multisampler and an AdvanceBio 300A 2.7 μm 4.6 × 300 mm (Agilent) SEC column. The SEC–MALS column was pre-equilibrated in buffer consistent with the individual experimental condition (50 mM Hepes [pH = 8.2], 200 mM NaCl, 5% [v/v] glycerol, and either NAD⁺ or NADH). The experiment was initiated with a 15 μl injection of 90 μM protein, and the column was run at room temperature and 0.25 ml/min. SEC–MALS data were collected as the protein migrated out of the SEC column on a Wyatt Neon DAWN ambient and analyzed using Astra Software, version 8.2. Concentrations were measured using both a 1260 Infinity II multiwavelength detector monitoring absorbance at 280 nm and the OptiLab Refractive Index Detector (Wyatt). DLS experiments were conducted with 5 μM to 1 mM enzyme, 1 mM to 2 mM coenzyme, and 1 mM to 2 mM substrate–product as indicated. DLS data were collected at 25 °C using a Wyatt microvolume disposable cuvette on a Wyatt DynaPro NanoStar II, with three data segments collected per sample with a 1-min incubation between each segment. DLS data were analyzed using Dynamics Software, version 8.2 (Waters, Wyatt Technology).

In total, 200 μg of EppA was injected onto a Yarra 3u SEC-2000 (Phenomenex; Torrance, CA, USA) size-exclusion column and eluted isocratically by EppA assay buffer. The 280 nm absorbance, laser light scattering, and differential refractive index were measured in tandem by a 280 nm UV detector (Agilent Technologies 1260 Infinity II), a DAWN HELEOS II 8+ (Wyatt Technology; Santa Barbara, CA, USA), and an Optilab T-rEX (Wyatt Technology; Santa Barbara, CA, USA), respectively. The molecular mass of EppA was calculated in ASTRA 7.1.4.8 (Wyatt Technology; Santa Barbara, CA, USA) by Zimm fitting measured light scattering intensities.