title: Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway.
abstract: Proteins frequently fold via folding intermediates that correspond to local minima on the conformational energy landscape. Probing the structure of the partially unfolded forms in equilibrium under native conditions can provide insight into the properties of folding intermediates. To elucidate the structures of folding intermediates of Escherichia coli dihydrofolate reductase (DHFR), we investigated transient partial unfolding of DHFR under native conditions. We probed the structure of a high-energy conformation susceptible to proteolysis (cleavable form) using native-state proteolysis. The free energy for unfolding to the cleavable form is clearly less than that for global unfolding. The dependence of the free energy on urea concentration (m-value) also confirmed that the cleavable form is a partially unfolded form. By assessing the effect of mutations on the stability of the partially unfolded form, we found that native contacts in a hydrophobic cluster formed by the F-G and Met-20 loops on one face of the central β-sheet are mostly lost in the partially unfolded form. Also, the folded region of the partially unfolded form is likely to have some degree of structural heterogeneity. The structure of the partially unfolded form is fully consistent with spectroscopic properties of the near-native kinetic intermediate observed in previous folding studies of DHFR. The findings suggest that the last step of the folding of DHFR involves organization in the structure of two large loops, the F-G and Met-20 loops, which is coupled with compaction of the rest of the protein.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, 47907.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Kasper
authors.forename: Pei-Fen
authors.initials: PF
authors.lastname: Liu
authors.forename: Chiwook
authors.initials: C
authors.lastname: Park
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D058767:Protein Unfolding; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
chemical_list: D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase
keywords: DHFR; folding intermediate; folding pathway; partially unfolded form; proteolysis