Folding mechanism of an extremely thermostable (ßα)(8)-barrel enzyme: a high kinetic barrier protects the protein from denaturation.
Biochemistry
; 51(16): 3420-32, 2012 Apr 24.
Article
em En
| MEDLINE
| ID: mdl-22455619
ABSTRACT
HisF, the cyclase subunit of imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima, is an extremely thermostable (ßα)(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 (ßα)(8)-barrel proteins.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Dobramento de Proteína
/
Thermotoga maritima
/
Aminoidrolases
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article