title: Folding mechanism of an extremely thermostable (βα)(8)-barrel enzyme: a high kinetic barrier protects the protein from denaturation.
abstract: HisF, the cyclase subunit of imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima, is an extremely thermostable (βα)(8)-barrel protein. We elucidated the unfolding and refolding mechanism of HisF. Its unfolding transition is reversible and adequately described by the two-state model, but 6 weeks is necessary to reach equilibrium (at 25 °C). During refolding, initially a burst-phase off-pathway intermediate is formed. The subsequent productive folding occurs in two kinetic phases with time constants of ~3 and ~20 s. They reflect a sequential process via an on-pathway intermediate, as revealed by stopped-flow double-mixing experiments. The final step leads to native HisF, which associates with the glutaminase subunit HisH to form the functional ImGPS complex. The conversion of the on-pathway intermediate to the native protein results in a 10(6)-fold increase of the time constant for unfolding from 89 ms to 35 h (at 4.0 M GdmCl) and thus establishes a high energy barrier to denaturation. We conclude that the extra stability of HisF is used for kinetic protection against unfolding. In its refolding mechanism, HisF resembles other (βα)(8)-barrel proteins.
authors.affiliation: Universität Regensburg, Institut für Biophysik und physikalische Biochemie, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors.forename: Linn
authors.initials: L
authors.lastname: Carstensen
authors.forename: Gabriel
authors.initials: G
authors.lastname: Zoldák
authors.forename: Franz-Xaver
authors.initials: FX
authors.lastname: Schmid
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sterner
mesh_terms: D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D013696:Temperature; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: C087227:imidazole glycerol phosphate synthase; D000619:Aminohydrolases