| Traditional stopped-flow methods for following the kinetic course of protein folding reactions are limited, in that substantial refolding occurs in the dead-time of mixing. A method of circumventing the mixing limitation is presented, based on the photochemical initiation of protein folding events. The method is based on the photolysis of small organic protecting groups, or “cage” compounds. These cage compounds are placed on side chains or within the main chain in a manner that may be expected to destabilize the native state. Photolysis of the cages rapidly generates a protein capable of refolding, without resorting to relatively slow mixing techniques.; The compounds that have been studied for this purpose are based on 3 ′,5′-dimethoxybenzoin (DMB). Esters of DMB are known to photolyze rapidly (on the picosecond timescale), generating the parent acid and the inert photoproduct 5,7-dimethoxy-2-phenylbenzofuran. In order for DMB cages to be used in folding studies, several derivatives were prepared. First, the linker 3′-carboxymethoxybenzoin was synthesized, which would allow the preparation of head-to-tail and head-to-side chain cyclized proteins. For proteins that have terminal residues that are far apart in the native state, cyclization would be expected to destabilize final structure. Photolysis of this cyclized form produces the linear polypeptide, which may then refold. Second, both DMB methoxy groups were replaced with carboxymethoxy groups, yielding a water-soluble cage. These cages may be selectively placed on the side chains of aspartic acid and glutamic acid, using solid-phase synthesis protocols. The water soluble DMB derivatives can disrupt the native structure through either sterics, or the introduction of a charge in the hydrophobic core of a protein. Each of these modified DMB groups has the same photolysis properties as the parent compound. Appropriately protected derivatives for each cage have been prepared for Fmoc solid-phase peptide synthesis, and the peptide synthesis protocols have been optimized. A series of caged polypeptides based on the GCN4 leucine zipper was examined by flash photolysis. One member of this series has a cage at a buried aspartate residue. This peptide is folded at neutral pH, but unfolds rapidly (700 ns) upon photolysis due charge repulsion between the internal carboxylates. |
