Yujie Chen

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.

Aggregation of α-synuclein is kinetically controlled by intramolecular diffusion

We hypothesize that the first step of aggregation of disordered proteins, such as α-synuclein, is controlled by the rate of backbone reconfiguration. When reconfiguration is fast, bimolecular association is not stable, but as reconfiguration slows, association is more stable and subsequent aggregation is faster. To investigate this hypothesis, we have measured the rate of intramolecular diffusion in α-synuclein, a protein involved in Parkinson’s disease, under solvent conditions that accelerate or decelerate aggregation. Using the method of tryptophan-cysteine (Trp-Cys) quenching, the rate of intramolecular contact is measured in four different loops along the chain length. This intrinsically disordered protein is highly diffusive at low temperature at neutral pH, when aggregation is slow, and compacts and diffuses more slowly at high temperature or low pH, when aggregation is rapid. Diffusion also slows with the disease mutation A30P. This work provides unique insights into the earliest steps of α-synuclein aggregation pathway and should provide the basis for the development of drugs that can prevent aggregation at the initial stage.

Ruggedness in the folding landscape of protein L

By exploring the folding pathways of the B1 domain of protein L with a series of equilibrium and rapid kinetic experiments, we have found its unfolded state to be more complex than suggested by two‐state folding models. Using an ultrarapid mixer to initiate protein folding within ∼2–4 microseconds, we observe folding kinetics by intrinsic tryptophan fluorescence and fluorescence resonance energy transfer. We detect at least two processes faster than 100 μs that would be hidden within the burst phase of a stopped‐flow instrument measuring tryptophan fluorescence. Previously reported measurements of slow intramolecular diffusion are commensurate with the slower of the two observed fast phases. These results suggest that a multidimensional energy landscape is necessary to describe the folding of protein L, and that the dynamics of the unfolded state is dominated by multiple small energy barriers.

Conformational properties of unfolded HypF-N

We have measured the intramolecular diffusion rate between distant residues in the aggregation-prone protein HypF-N under various denaturing conditions. Using the method of cysteine quenching of the tryptophan triplet state, we find that intramolecular diffusion remains roughly constant at high concentrations of denaturant (2-6 M GdnHCl) and slows down at low concentrations of denaturant, but the decrease is not uniform throughout the chain. Extrapolation of these measurements to 0 M GdnHCl gives D approximately 10(-7) cm(2) s(-1), about 1 order of magnitude lower than unstructured peptides and at least 2 orders of magnitude higher than well-behaved proteins. This suggests that there is a dynamic range of conformational reorganization within which partially unfolded states are prone to aggregation.

A general polymer model of unfolded proteins under folding conditions.

There is increasing evidence that a polypeptide chain in solvent conditions that favor folding may have transient structure and is significantly more compact than a fully denatured chain. However, there is no sequence-dependent model to capture such interactions. In this work, we present a simple and computationally inexpensive model based on a wormlike chain with excluded volume. The probability distribution of millions of such chains is reweighted to bias compact conformations in which residues of similar hydrophobicity are located near each other. This model, which has one adjustable parameter, is fit to measured values of intramolecular contact formation, which has been shown to be extremely sensitive to various models of intrachain distances. We show that under various denaturant concentrations, there is good convergence of the model for several different sequences with a wide range of dynamics. We also show that this model quantitatively predicts paramagnetic resonance enhancement (PRE) measurements with no adjustable parameters. Therefore a simple, probabilistic model that accounts for sequence-specific interactions may give a more realistic starting point for predictions of protein folding.

Combined Force Ramp and Equilibrium High-Resolution Investigations Reveal Multi-Path Heterogeneous Unfolding of Protein G

Over the past two decades, one of the standard models of protein folding has been the two-state model, in which a protein only resides in the folded or fully unfolded states with a single pathway between them. Recent advances in spatial and temporal resolution of biophysical measurements have revealed beyond-two-state complexity in protein folding, even for small, single-domain proteins. In this work, we used high-resolution optical tweezers to investigate the folding/unfolding kinetics of the B1 domain of immunoglobulin-binding protein G (GB1), a well-studied model system. Experiments were performed for GB1 both in and out of equilibrium using force spectroscopy. When the force was gradually ramped, simple single-peak folding force distributions were observed, while multiple rupture peaks were seen in the unfolding force distributions, consistent with multiple force-dependent parallel unfolding pathways. Force-dependent folding and unfolding rate constants were directly determined by both force-jump and fixed-trap measurements. Monte Carlo modeling using these rate constants was in good agreement with the force ramp data. The unfolding rate constants exhibited two different behaviors at low vs high force. At high force, the unfolding rate constant increased with increasing force, as previously reported by high force, high pulling speed force ramp measurements. However, at low force, the situation reversed and the unfolding rate constant decreased with increasing force. Taken together, these data indicate that this small protein has multiple distinct pathways to the native state on the free energy landscape.

Diffusion of Unfolded Acyl-Coenzyme A-Binding Protein Over a Complete Range of Denaturant

The unfolded states of acyl-coenzyme A-binding protein (ACBP) were studied from the point of view of diffusion dynamics. Using the method of Trp/Cys contact quenching, we monitored the intramolecular diffusion of the unfolded chain over a wide range of denaturant in both equilibrium and using a novel microfluidic mixer to capture dynamics before folding. Theoretically, both a worm-like chain model and molecular dynamics (MD) have been used to generate loop terminal distance distributions required for data analysis. MD simulation also provides a direct comparison to measured rates through mean squared displacement over time. We observed a deep compaction of protein conformation from 6M GuHCL to 0.2M GuHCL resulting in a 100-fold decrease of intramolecular diffusion coefficient. This protein, however, is still shown to be more diffusive than protein L in physiological conditions, suggesting a strong sequence dependence of intramolecular diffusion.

Modeling Drkn Sh3 Domain Using Sequence Specific Wormlike Chain Model

Though the wormlike chain (WLC) model has successfully described the statistical properties of fully denatured polypeptides, the lack of sequence details and attractive forces made it less successful in describing unfolded states in folding conditions. To cover the limitation while keeping the models efficient feature, we have developed a sequence specific wormlike chain model. Computationally, secondary structure constraints from the secondary chemical shift measurements of drkN are integrated into the construction of each wormlike chain. Then the probability distribution is reweighted to bias compact conformations in which residues of similar hydrophobicity are located near each other. This model has been tested on two mutants (C2 and C60) of the Drosophila drk N-terminal (drkN) SH3 domain. drkN exists in approximately 1:1 equilibrium between folded and unfolded state in water, which gives us the opportunity to monitor the contact quenching of tryptophan 36 by either cysteine under all denaturing conditions. The experimental results exhibit a relatively slow kinetics, which implies slow intramolecular diffusion. The reweighted pairwise distance distributions are also compared to the Paramagnetic Relaxation Enhancement (PRE) data for drkN.