Conformational stabilization and rapid labeling of a 29-residue peptide by a small molecule reaction partner.

A 29-residue peptide (MP01) - identified by in vitro selection for reactivity with a small molecule perfluoroaromatic - was modified and characterized using experimental and computational techniques, with the goal of understanding the molecular basis of its reactivity. These studies identified a six amino acid point mutant (MP01-Gen4) that exhibited a reaction rate constant of 25.8 ± 1.8 M-1 s-1 at pH 7.4 and room temperature, approximately two orders of magnitude greater than its progenitor sequence and three orders of magnitude greater than background cysteine reactivity. MP01-Gen4 appeared conformationally dynamic and exhibited several properties reminiscent of larger protein molecules, including denaturant-sensitive structure and reactivity. We believe the majority of the reaction rate enhancement can be attributed to interaction of MP01-Gen4 with the perfluoroaromatic probe, which was found to stabilize a helical conformation of both MP01-Gen4 and non-reactive (Cys-to-Ser) or (Cys-to-Ala) variants. These findings demonstrate the ability of dynamic peptides to access protein-like reaction mechanisms, and the potential of perfluoroaromatic functionality to stabilize small peptide folds.