title: Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain.
abstract: Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (∼10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured state. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein-protein interactions.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University , BMC Box 582, SE-75123 Uppsala, Sweden.
authors.forename: Jakob
authors.initials: J
authors.lastname: Dogan
authors.affiliation: Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW, United Kingdom.
authors.forename: Angelo
authors.initials: A
authors.lastname: Toto
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University , BMC Box 582, SE-75123 Uppsala, Sweden.
authors.forename: Eva
authors.initials: E
authors.lastname: Andersson
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" Sapienza, University of Rome , 00185 Rome, Italy.|Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW, United Kingdom.
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University , BMC Box 582, SE-75123 Uppsala, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
mesh_terms: D050882:CREB-Binding Protein / Q000737:chemistry*; D002942:Circular Dichroism; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet
chemical_list: D050882:CREB-Binding Protein