title: Slow unfolded-state structuring in Acyl-CoA binding protein folding revealed by simulation and experiment.
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.
authors: Department of Chemistry, Stanford University, Stanford, California 94305-5080, United States.
authors: Vincent A
authors: VA
authors: Voelz
authors: Marcus
authors: M
authors: Jäger
authors: Shuhuai
authors: S
authors: Yao
authors: Yujie
authors: Y
authors: Chen
authors: Li
authors: L
authors: Zhu
authors: Steven A
authors: SA
authors: Waldauer
authors: Gregory R
authors: GR
authors: Bowman
authors: Mark
authors: M
authors: Friedrichs
authors: Olgica
authors: O
authors: Bakajin
authors: Lisa J
authors: LJ
authors: Lapidus
authors: Shimon
authors: S
authors: Weiss
authors: Vijay S
authors: VS
authors: Pande
mesh_terms: D000818:Animals; D002417:Cattle; D026261:Diazepam Binding Inhibitor / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D057927:Hydrophobic and Hydrophilic Interactions; D008390:Markov Chains; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D055550:Protein Stability; D058767:Protein Unfolding
chemical_list: D026261:Diazepam Binding Inhibitor