Figure 6.

(A) Structures of nonfluorescent analogues of biotin (19–22) used in competition binding assays. (B) Quantification of competitive inhibitory constants (K_i) of 19–22 for SA complexed with 18 (175 nM for SA and 25 nM for 18) by Trp-FRET. Tryptophan residues were excited at 295 nM, and FRET was measured at 460 nm. Values were corrected to account for fluorescence quenching upon binding as described in Experimental Section. [SA] was based on monomeric protein. Half-maximal inhibitory concentrations (IC₅₀) were calculated using a log(inhibitor) vs response model (GraphPad Prism 6.0), and IC₅₀ values were converted to K_i values. (C) Evaluation of direct binding of 20 to SA using ITC. Compound 20 was titrated into [SA] in PBS (pH 7.4), and thermodynamic parameters and K_d values were calculated using the Origin software. [SA in sample cell] = 20 μM and [20 in syringe] = 250 μM.