Folding mechanism of an extremely thermostable (βα)₈-barrel enzyme : a high kinetic barrier protects the protein from denaturation

Title data

Carstensen, Linn ; Zoldák, Gabriel ; Schmid, Franz X. ; Sterner, Reinhard:
Folding mechanism of an extremely thermostable (βα)₈-barrel enzyme : a high kinetic barrier protects the protein from denaturation.
In: Biochemistry. Vol. 51 (2012) Issue 16 . - pp. 3420-3432.
ISSN 1520-4995
DOI: https://doi.org/10.1021/bi300189f

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Abstract in another language

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.