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

Authors

  • Angela Morrone,

    1. Istituto Pasteur-Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, Piazzale A. Moro 5, 00185 Rome, Italy
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  • Rajanish Giri,

    1. Istituto Pasteur-Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, Piazzale A. Moro 5, 00185 Rome, Italy
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  • Maurizio Brunori,

    1. Istituto Pasteur-Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, Piazzale A. Moro 5, 00185 Rome, Italy
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  • Stefano Gianni

    Corresponding author
    1. Istituto Pasteur-Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, Piazzale A. Moro 5, 00185 Rome, Italy
    • Istituto Pasteur-Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A., Rossi Fanelli”, Sapienza Università di Roma, Piazzale A. Moro 5, 00185 Rome, Italy
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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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