PylB proteolytic degradation assays

Solid α-chymotrypsin (Sigma-Aldrich, cat. # C3142) stock solution was first prepared by dissolving the enzyme in 1 mM HCl to a concentration of 0.05 mg/mL; chymotrypsin solutions were stored for up to 5 days following resuspension. Purified and His-tag-cleaved PylB samples were each concentrated to a uniform concentration of 1 mg/mL,120 µL aliquots of each sample were added to a separate tube, supplemented with 10 mM CaCl₂, and allowed to warm to room temperature. Tubes were then pre-labeled for each time point for each sample, and 6.7 µL SDS-PAGE loading dye was added to each tube. Prior to initiating digestion, an undigested 24 µL aliquot of each sample was removed and mixed with loading dye and 2.8 µL of 1 mM HCl (termed the 0-min sample). To the remaining 96 µL of each protein sample, 9.6 µL of chymotrypsin solution was then added, and post-digestion time was recorded. Aliquots were taken from each sample at the following time-points: 30 s, 1 min, 2 min, 5 min, 10 min, and 15 min. Upon being removed from the digestion reaction, each aliquot was mixed with loading dye in a separate tube, and immediately placed into a heat-block preheated to 98 °C. Each sample was boiled for 15 min, chilled on ice for at least 2 min, and a 4-8 µL aliquot was then run on a pre-cast SDS-PAGE gel (Bio-Rad cat. #4568096) before staining with Coomassie blue dye. As a standard, 4 µL of BLUEstain 2 protein ladder (GOLDBIO cat. #P008-500) was also loaded on each gel. Stained gels were imaged, and PylB bands were quantified in each sample using ImageJ; band intensities were converted into protein concentration based on observed intensities of ladder samples, as the concentration of the ladder was provided by the supplier. At each time point, the amount of enzyme product (degraded PylB) produced was calculated by subtracting the PylB concentration at that time point from the initial PylB concentration. From these data, catalytic constant (k_cat) values for chymotrypsin degradation of each PylB variant were calculated using Graphpad Prism 8.