1 research outputs found
Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment
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