Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment
Vincent A. VoelzMarkus JägerShuhuai YaoYujie ChenLi ZhuSteven A. WaldauerGregory R. BowmanMark S. FriedrichsOlgica BakajinLisa J. LapidusShimon WeissVijay S. Pande
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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.Keywords:
Folding (DSP implementation)
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The first part of this paper contains an overview of protein structures, their spontaneous formation ("folding"), and the thermodynamic and kinetic aspects of this phenomenon, as revealed by in vitro experiments. It is stressed that universal features of folding are observed near the point of thermodynamic equilibrium between the native and denatured states of the protein. Here the "two-state" ("denatured state" "native state") transition proceeds without accumulation of metastable intermediates, but includes only the unstable "transition state". This state, which is the most unstable in the folding pathway, and its structured core (a "nucleus") are distinguished by their essential influence on the folding/ unfolding kinetics. In the second part of the paper, a theory of protein folding rates and related phenomena is presented. First, it is shown that the protein size determines the range of a proteins folding rates in the vicinity of the point of thermodynamic equilibrium between the native and denatured states of the protein. Then, we present methods for calculating folding and unfolding rates of globular proteins from their sizes, stabilities and either 3D structures or amino acid sequences. Finally, we show that the same theory outlines the location of the protein folding nucleus (i.e., the structured part of the transition state) in reasonable agreement with experimental data.
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Guanidine
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For apparently two-state proteins, we found that the size (number of folded residues) of a transition state is mostly encoded by the topology, defined by total contact distance (TCD) of the native state, and correlates with its folding rate. This is demonstrated by using a simple procedure to reduce the native structures of the 41 two-state proteins with native TCD as a constraint, and is further supported by analyzing the results of eight proteins from protein engineering studies. These results support the hypothesis that the major rate-limiting process in the folding of small apparently two-state proteins is the search for a critical number of residues with the topology close to that of the native state.
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Guided by the recent success of empirical model predicting the folding rates of small two-state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L(2/3), and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs_CO = CO x L, is able to predict rather accurately folding rates for both two-state and multistate folding proteins, as well as short peptides, and that this Abs_CO scales with the protein chain length as L(0.70 +/- 0.07) for the totality of studied single-domain proteins and peptides.
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Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier. This protein is very stable and topologically simple, needing merely to “zipper up,” while having an extrapolated folding rate of k f = 2 × 10 5 s -1 . These properties make it likely to attain the elusive “downhill folding” limit, at which a series of intermediates can be characterized. To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods. The stability and its denaturant dependence for the hydrogen bonds in the central part of protein equal the values calculated for whole-molecule unfolding. Like-wise, their closing and opening rates indicate that these hydrogen bonds are broken and reformed in a single cooperative event representing the folding transition from the fully unfolded state to the native state. Additionally, closing rates for these hydrogen bonds agree with the extrapolated barrier-limited folding rate observed near the melting transition. Therefore, even in the absence of denaturant, where Δ G eq ≈ -6 kcal·mol -1 (1 cal = 4.18 J) and τ f ≈ 6 μs, folding remains cooperative and barrier-limited. Given that this prime candidate for downhill folding fails to do so, we propose that protein folding will remain barrier-limited for proteins that fold cooperatively.
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A buried ionizable residue can have a drastic effect on the stability of a native protein, but there has been only limited investigation of how burial of an ionizable residue affects the kinetics of protein folding. In this study, the effect of burial of ionizable residues on the thermodynamics and kinetics of folding and unfolding of monellin has been investigated. The stability of wild-type (wt) monellin is known to decrease with an increase in pH from 4 to 10. The Glu24 → Ala mutation makes the stability of the resultant E24A mutant protein independent of pH in the range from 4 to 8. An additional mutation, Cys42 → Ala, results in the stability becoming independent of pH in the range from 4 to 10. Like the wt protein, E24A folds via very fast, fast, and slow folding pathways. Compared to that of the wt protein, the rate of slow folding pathway of E24A is ~7-fold faster, the rate of fast folding pathway is ~1.5-fold faster, while the rate of very fast folding pathway is similar. E24A unfolds ~7-fold slower than the wt. The extent of stabilization of the transition state (TS) observed for the slow pathway of refolding and for unfolding is the same, indicating that unfolding occurs via the TS populated on the slow pathway of refolding. The stabilization of the TS of folding (1.1 kcal mol(-1)) is less than that of the native state (2.3 kcal mol(-1)) of E24A, indicating that structure has only partially formed in the vicinity of Glu24 in the TS of folding.
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Energy landscape
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