Dynapro nanostar 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).

We characterized
the molecular weight of the CD by MALDI-MS and SDS-PAGE. For MALDI-MS,
the different CD preparations were suspended in a matrix of sinapinic
acid before analysis (Shimadzu Biotech, AXIMA Confidence). For SDS-PAGE,
all CD preparations were resuspended in water at 2 mg/mL, and 18.75
μL was mixed with 6.25 μL of 80% glycerol and separated
using 4–20% SDS-PAGE (BioRad; #4561093). We compared the band
positions relative to those of a protein ladder standard to determine
the molecular weight of the CD preparations. To identify the CD-biotin
and CD-Cy5 conjugates, gel images were analyzed at 488 nm (CD) and
647 nm (Cy5) using a gel documentation system (BioRad, Chemidoc MP
Imaging System). To determine the size of the CD, we used dynamic
light scattering (DLS) and transmission electron microscopy (TEM).
DLS was performed using a DynaPro NanoStar II instrument from Wyatt
Technology. For TEM, 7 μL of a 2 mg/mL CD suspension was dried
onto a Formvar-coated copper grid. CD images were then collected by
using a JEOL JEM-1400flash microscope.

The Wyatt DynaPro Nanostar II instrument was employed for DLS measurements. This instrument uses light scattering at 658 nm and a scattering angle of 90°. Protein samples of 20 μL in 50 mM Tris-HCl buffer (pH 7.5) containing 50 mM KCl, 10 mM MgCl₂, 1 mM DTT, and 20% glycerol were loaded in a small disposable cuvette for each measurement. The measurements were carried out at 20 °C.