title: 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).
authors.affiliation: 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.
authors.forename: Angela
authors.initials: A
authors.lastname: Morrone
authors.forename: Rajanish
authors.initials: R
authors.lastname: Giri
authors.forename: Maurizio
authors.initials: M
authors.lastname: Brunori
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
mesh_terms: D000818:Animals; D050882:CREB-Binding Protein / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014508:Urea / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D014508:Urea; D050882:CREB-Binding Protein