Title
Activation barrier-limited folding and conformational sampling of a dynamic protein domain
Author
Jakob Dogan, Angelo Toto, Eva Andersson, Stefano Gianni, Per Jemth
Year
2016
Journal
Biochemistry
Abstract
Folding reaction mechanisms of globular protein domains have been extensively studied both by experiment and simulation and found to be highly concerted chemical reactions in which numerous non-covalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins since their folding can usually only be studied in conjunction with binding to a ligand. We have here 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 NMR data showing that NCBD exists in two folded and one more denatured conformation at equilibrium and, thus, that the folding mechanism is three-state. 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. Since the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein-protein interactions.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Protein folding, Kinetics, Biochemistry