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Reassessing the folding of the KIX domain: Evidence for a two‐state mechanism

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).