Abstract 1512 Quenching of dynamic shifts in the tryptophan fluorescence of an acyl protein thioesterase

FTT258 from Francisella tularensis is a bacterial homologue of human acyl protein thioesterases (hAPTs). APTs catalyze the reversible removal of S-acylated palmitoylations added post-translationally to various cellular proteins including key cancer signaling proteins. In this study, we used quenching of the intrinsic fluorescence of three tryptophan residues on FTT258 to monitor shifts in the structural dynamics of FTT258 upon lipid and membrane binding. Changes in the tryptophan fluorescence were measured in response to increasing concentrations of irreversible/reversible serine hydrolase inhibitors and micelle forming detergents that mimic lipid binding. Using this combination of inhibitors and detergents, we monitored their effects on the tryptophan fluorescence of wild-type FTT258 and FTT258 variants with tryptophan residues converted to phenylalanine. Control samples of a soluble form of tryptophan, N-Acetyl-L-tryptophanamide (NATA), were used to remove background fluorescence due to the inner filter effect. The amount of tryptophan fluorescence emission was monitored over time to follow dynamic shifts in FTT258 structure upon inhibitor and detergent binding and release. Changes in tryptophan fluorescence were then correlated with locations of tryptophan substitution to map shifts in protein structure to specific dynamic loops and exposed patches on the FTT258 structure, finding that distinct structural faces control substrate versus inhibitor binding. Results with FTT258 will eventually be transferred onto the structure of human APTs to provide evidence for their structural dynamics and how it controls their substrate selectivity. Funded by NSF MCB-1812971.