Identification of an essential backbone amide bond in the folding and stability of a multimeric enzyme

Here we utilize a total chemical synthesis strategy and a mass spectrometry -based, combinatorial chemistry approach to identify key molecular interact ions that contribute to the folding and stability of a model multimeric enz yme, 4-oxalocrotonate tautomerase (4OT). 4OT is a 41 kDa bacterial enzyme c omposed of six identical 62 amino acid subunits. A total of 16 different 4O T analogues containing various natural and unnatural N-terminal modificatio ns were prepared by total chemical synthesis and then characterized by cata lytic activity, size exclusion chromatography (SEC), and circular dichroism (CD) spectroscopy. The results of our mutational studies indicate that bac kbone-backbone hydrogen-bonding interactions involving the amide bond betwe en Pro1 and Ile2 in 4OT's 62 amino acid polypeptide chain play an important role in specifying the conformation of this enzyme's native folded state. These results provide the first evidence that backbone-backbone hydrogen-bo nding interactions can play such a major role in specifying the native stat e of a protein.