title: Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme.
abstract: Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Hermann-Herder-Strasse 7, Universität Freiburg, D-79104, Germany.
authors.forename: O
authors.initials: O
authors.lastname: von Ahsen
authors.forename: J H
authors.initials: JH
authors.lastname: Lim
authors.forename: P
authors.initials: P
authors.lastname: Caspers
authors.forename: F
authors.initials: F
authors.lastname: Martin
authors.forename: H J
authors.initials: HJ
authors.lastname: Schönfeld
authors.forename: J
authors.initials: J
authors.lastname: Rassow
authors.forename: N
authors.initials: N
authors.lastname: Pfanner
mesh_terms: D000818:Animals; D002384:Catalysis; D018834:Chaperonin 60 / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D005493:Folic Acid Antagonists / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D008024:Ligands; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D009492:Neurospora crassa / Q000201:enzymology; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D015003:Yeasts / Q000201:enzymology
chemical_list: D018834:Chaperonin 60; D005493:Folic Acid Antagonists; D008024:Ligands; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D019286:Endopeptidase K; D019696:Peptidylprolyl Isomerase; D008727:Methotrexate