Sidechain behavior during folding of Eschericha coli dihydrofolate reductase studied by stopped-flow (19)F NMR

The information contained in the amino acid sequence of many proteins is sufficient to direct the formation of the correct, biologically active tertiary structure. Yet the mechanism by which this occurs remains unclear. Structural information about events which occur during protein folding has been difficult to obtain; information about unfolding, and about changes in sidechain environment and mobility during protein folding is particularly limited.;This research has focused on changes in the sidechain environment of E. coli dihydrofolate reductase (DHFR) upon ligand addition; as a function of urea concentration at equilibrium; and upon unfolding or refolding in the presence or absence of ligands using stopped-flow NMR spectroscopy. These results are compared with those obtained by circular dichroism and fluorescence spectroscopies.;E. coli DHFR contains five tryptophans distributed throughout the molecule in different secondary structural elements. The experimental strategy employed has been to incorporate 6-;At equilibrium, Trp22 exchanges rapidly between native and unfolded environments, while the remaining regions exchange slowly with distinct native and unfolded resonances. Kinetic NMR studies reveal that the majority of the protein unfolds via an intermediate which contains secondary structure but in which the sidechains are disordered. Under the conditions used to study refolding by stopped-flow NMR, the unfolded form and an early intermediate inter-convert, with a substantial amount of the protein remaining unfolded. These NMR studies reveal that solvent exclusion (detected by fluorescence) and secondary structure formation (detected by circular dichroism) monitor the disappearance of unfolded environment rather than the appearance of native-like side chain environment. For the apo protein, formation of native sidechain environment is the final step in folding with different regions of the protein forming native-like environment at different rates. In the presence of NADP...