Carstensen, Linn and Zoldak, Gabriel and Schmid, Franz-Xaver and Sterner, Reinhard (2012) Folding Mechanism of an Extremely Thermostable (beta alpha)(8)-Barrel Enzyme: A High Kinetic Barrier Protects the Protein from Denaturation. BIOCHEMISTRY, 51 (16). pp. 3420-3432. ISSN 0006-2960,
Full text not available from this repository. (Request a copy)Abstract
HisF, the cyclase subunit of imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima, is an extremely thermostable (beta alpha)(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 degrees 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 similar to 3 and similar to 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 (beta alpha)(8)-barrel proteins.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | TIM BARREL PROTEIN; INDOLE-3-GLYCEROL PHOSPHATE SYNTHASE; EXCHANGE MASS-SPECTROMETRY; GLOBAL ANALYSIS HIGHLIGHTS; ALPHA-SUBUNIT; TRYPTOPHAN SYNTHASE; THERMOTOGA-MARITIMA; ESCHERICHIA-COLI; TRP SYNTHASE; OFF-PATHWAY; |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 18 May 2020 05:43 |
| Last Modified: | 18 May 2020 05:43 |
| URI: | https://pred.uni-regensburg.de/id/eprint/18874 |
Actions (login required)
 |
View Item |
