Synthesis and physicochemical characterization of (6S)-5-formiminotetrahydrofolate; a reference standard for metabolomics.

The one-carbon carrier of the formate oxidation level derived from the interaction of tetrahydrofolate and formiminoglutamate, which has been tentatively identified as 5-formiminoltetrahydrofolate, has been prepared by chemical synthesis. Treatment of a solution of (6S)-tetrahydrofolate in aqueous base with excess ethyl formimidate in the presence of anti-oxidant under anaerobic conditions afforded a gummy solid which, based on mass spectral analysis, conformed to a monoformimino derivative of tetrahydrofolate. Further physicochemical characterization by validated methods strongly suggested that the product of chemical synthesis was identical to the enzymatically produced material and that it was, in fact, (6S)-5-formiminotetrahydrofolate. Conditions and handling methods toward maintaining the integrity of this highly sensitive compound were identified and are described, as is analytical methodology, useful for research studies using it.

Guanidine-induced equilibrium unfolding of a homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT).

4-Oxalocrotonate tautomerase (4-OT) is a bacterial enzyme that is comprised of 6 identical 62 amino acid subunits. The 4-OT enzyme is an attractive model system in which to study the interrelationship between protein folding, subunit assembly, and catalytic function. Here we report on the GuHCl-induced equilibrium unfolding properties of wild-type 4-OT using catalytic activity measurements and using far-UV circular dichroism (CD) spectroscopy. We demonstrate that the unfolding of wild-type 4-OT in 50 mM phosphate buffers containing 6 M GuHCl is reversible at pHs 6.0, 7.4, and 8.5; and we find that there is both an enzyme concentration dependence and a pH dependence to the equilibrium unfolding properties of 4-OT. Our data suggests that the GuHCl-induced unfolding of 4-OT in 50 mM phosphate buffer at pH 8.5 can be modeled as a two-state process involving folded hexamer and unfolded monomer. On the basis of this model, we determined a free-energy value for the unfolding of 4-OT at pH 8.5 to be 68.7 +/- 3.2 kcal/mol under standard state conditions (1 M hexamer). In 50 mM phosphate buffers at pHs 6.0 and 7.4, only the catalytic activity denaturation curves are consistent with a two-state folding mechanism. At the lower pHs the far-UV-CD transitions are not well described by a two-state model. Our results at pHs 6.0 and 7.4 suggest that intermediate state(s) are populated in the equilibrium unfolding reaction at these lower pHs and that these intermediate state(s) have some helical content but no measurable catalytic activity.

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 studying protein folding reactions is described, and its initial application to study the folding/unfolding reaction of a homo-hexameric enzyme 4-oxalocrotonate (4OT) is reported. This new approach involves the application of total chemical synthesis to prepare protein analogues that contain a photoreactive amino acid site-specifically incorporated into their primary amino acid sequence. To this end, a photoreactive amino acid-containing analogue of 4OT in which Pro-1 was replaced with p-benzoyl-l-phenylalanine (Bpa) was prepared. This analogue can be used to map structurally specific protein-protein interactions in 4OT's native folded state. These photocrosslinking studies and peptide mapping results with (PlBpa)4OT indicate that this construct is potentially useful for probing the structural properties of equilibrium and kinetic intermediates in 4OT's folding reaction.