Slow unfolded-state structuring in Acyl-CoA binding protein folding revealed by simulation and experiment.
J Am Chem Soc
; 134(30): 12565-77, 2012 Aug 01.
Article
em En
| MEDLINE
| ID: mdl-22747188
ABSTRACT
Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A binding protein (ACBP), a two-state folder (folding time ~10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenesis, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond time scale. These studies, along with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (~100 µs) time scale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov state model (MSM) of the ACBP folding reaction, constructed from over 30 ms of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure, and kinetics consistent with experiment but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate but rather to a more heterogeneous and slow acquisition of unfolded-state structure.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Dobramento de Proteína
/
Inibidor da Ligação a Diazepam
Tipo de estudo:
Health_economic_evaluation
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
J Am Chem Soc
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos