The effects of altering the gateway base on the affinity and time-dependent change in affinity of SPN2 for DFHBI and PFP-DFHBI. (A) Affinities after 10 min incubation for PFP-DFHBI in buffer SX of SPN2 variants in position 71 as identified in the x-axis labels. (B) Comparison of split SPN2 (spSPN2) and spSPN2 lacking a purine at position 71 (spSPN2-Apu) for their affinities for DFHBI and PFP-DFHBI after 10 min incubation (open bars) and 3 h incubation at 23°C (hatched bars) in buffer IC. (C) Time dependency of the affinities in buffer SX of SPN2 variants as identified in the legend. All time dependencies are for binding to PFP-DFHBI except the one identified as SPN2 (DFHBI). The glycosidic bond is represented by the squiggle in each chemical structure. Each data set is from a titration performed as a single experiment and identified with a different color. Each titration was performed independently two to four times with duplicate samples in each experiment. Each data point is the result of a fitted data set, and each color represents a set of fits for all data sets in a single experiment. The average fitting (R2) for all experiments was 0.98.
