title: Kinetic analysis of R67 dihydrofolate reductase folding: from the unfolded monomer to the native tetramer.
abstract: R67 dihydrofolate reductase (DHFR) is a homotetrameric enzyme. Its subunit has a core structure consisting of five antiparallel beta-strands that form a compact beta-barrel. Our interest was to describe the molecular mechanism of the complete folding pathway of this beta-sheet protein, focusing on how the oligomerization steps are coordinated with the formation of secondary and tertiary structures all along the folding process. The folding kinetics of R67 dihydrofolate reductase into dimers at pH 5.0 were first examined by intrinsic tryptophan fluorescence, fluorescence energy transfer, and circular dichroism spectroscopy. The process was shown to consist of at least four steps, including a burst, a rapid, a medium, and a slow phase. Measurements of the ellipticity at 222 nm indicated that about 50% of the total change associated with refolding occurred during the 4 ms dead time of the stopped-flow instrument, indicating a substantial burst of secondary structure. The bimolecular association step was detected using fluorescence energy transfer and corresponded to the rapid phase. The slow phase was attributed to a rate-limiting isomerization of peptidyl-prolyl bonds involving 15% of the unfolded population. A complete folding pathway from the unfolded monomer to the native tetramer was proposed and an original model based upon the existence of early partially folded monomeric intermediates, rapidly stabilized in a dimeric form able to self-associate into the native homotetramer was formulated. The rate constants of these various steps were determined by fitting the kinetic traces to this model and supported our mechanistic assumptions.
authors.affiliation: Unité de Repliement et Modélisation des Protéines, CNRS URA 2185, Institut Pasteur, 28 rue du Dr. Roux 75724 Paris Cédex 15, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Bodenreider
authors.forename: Nicolas
authors.initials: N
authors.lastname: Kellershohn
authors.forename: Michel E
authors.initials: ME
authors.lastname: Goldberg
authors.forename: Annick
authors.initials: A
authors.lastname: Méjean
mesh_terms: D002942:Circular Dichroism; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011815:R Factors / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D019791:Guanidine