Reassessing the folding of the KIX domain: evidence for a two-state mechanism.

Abstract : The debate about the presence and role of intermediates in the folding of proteins has been a critical issue, especially for fast folders. One of the classical methodologies to identify such metastable species is the "burst-phase analysis," whereby the observed signal amplitude from stopped-flow traces is determined as a function of denaturant concentration. However, a complication may arise when folding is sufficiently fast to jeopardize the reliability of the stopped-flow technique. In this study, we reassessed the folding of the KIX domain from cAMP Response Element-Binding (CREB)-binding protein, which has been proposed to involve the formation of an intermediate that accumulates in the dead time of the stopped flow. By using an in-house-built capillary continuous flow with a 50-μs dead time, we demonstrate that this intermediate is not present; the problem arose because of the instrumental limitation of the standard stopped flow to assess very fast refolding rate constants (e.g., ≥ 500 s⁻¹).
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Angela Morrone, Rajanish Giri, Maurizio Brunori, Stefano Gianni. Reassessing the folding of the KIX domain: evidence for a two-state mechanism.. Protein Science, Wiley, 2012, 21 (11), pp.1775-9. ⟨10.1002/pro.2159⟩. ⟨pasteur-00960069⟩

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