Mapping tertiary interactions in protein folding reactions: a novel mass spectrometry- and chemical synthesis-based approach

A novel mass spectrometry- and chemical synthesis-based approach for studyi ng protein folding reactions is described, and its initial application to s tudy the folding/unfolding reaction of a homo-hexameric enzyme 4-oxalocroto nate (4OT) is reported. This new approach involves the application of total chemical synthesis to prepare protein analogues that contain a photoreacti ve amino acid site-specifically incorporated into their primary amino acid sequence. To this end, a photoreactive amino acid-containing analogue of 4O T in which Pro-1 was replaced with p-benzoyl-l-phenylalanine (Bpa) was prep ared. This analogue can be used to map structurally specific protein-protei n interactions in 4OT's native folded state. These photocrosslinking studie s and peptide mapping results with (P1Bpa)4OT indicate that this construct is potentially useful for probing the structural properties of equilibrium and kinetic intermediates in 4OT's folding reaction.