Fast Folding Research Articles

A “slow” protein folds quickly in the end

Until just a few years ago, it was not clear whether it would be possible to fold proteins using all-atom molecular dynamics simulations with explicit solvent molecules, despite the insights that these simulations had yielded into other biological problems. This was not just because of the computational challenge of reaching folding time scales, typically microseconds to seconds or longer, but also because it was not generally accepted that the empirical energy functions (“force fields”) used were sufficiently accurate to locate the folded state as a global free-energy minimum. This has changed dramatically in the last 2 y, with the development by Shaw and coworkers of a special-purpose supercomputer, Anton, capable of running biomolecular MD simulations on a microsecond or even millisecond timescale (1). This group showed that, with only minor adjustments to existing force fields (2⇓–4), it was possible to fold 12 small, “fast-folding” proteins (1), which adopt their native structure in microseconds. At the time, it was still not clear, however, whether it would be possible to do the same for larger, slower-folding proteins (5, 6). In PNAS, Piana et al. report simulations of ubiquitin folding, which occurs in milliseconds (7). Their results not only extend the computationally accessible time scale for calculating equilibrium folding trajectories by 2–3 orders of magnitude but have a number of implications that can only be deduced from a comparison of fast and slow folding proteins.

Native states of fast-folding proteins are kinetic traps.

It has been suggested that the native state of a protein acts as a kinetic hub that can facilitate transitions between nonnative states. Using recently developed tools to quantify mediation probabilities ("hub scores"), we quantify hub-like behavior in atomic resolution trajectories for the first time. We use a data set of trajectory ensembles for 12 fast-folding proteins previously published by D. E. Shaw Research (Lindorff-Larsen, K.; et al. How Fast-Folding Proteins Fold. Science2011, 334, 517) with an aggregate simulation time of over 8.2 ms. We visualize the free-energy landscape of each molecule using configuration space networks, and show that dynamic quantities can be qualitatively understood from visual inspection of the networks. Modularity optimization is used to provide a parameter-free means of tessellating the network into a group of communities. Using hub scores, we find that the percentage of trajectories that are mediated by the native state is 31% when averaged over all molecules, and reaches a maximum of 52% for the homeodomain and chignolin. Furthermore, for these mediated transitions, we use Markov models to determine whether the native state acts as a facilitator for the transition, or as a trap (i.e., an off-pathway detour). Although instances of facilitation are found in 4 of the 12 molecules, we conclude that the native state acts primarily as a trap, which is consistent with the idea of a funnel-like landscape.

Atomic-level description of ubiquitin folding.

Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins.

Thermodynamic theory of nonlinear ultrasound in soft biological tissue

A new theoretical model of ultrasound propagation in soft biological media is presented based on an extended thermodynamics formalism. The long-standing experimental conjecture claiming that a continuous distribution of internal degrees of freedom can be used to model ultrasound in biological media is given theoretical justification. A strategy to derive a well-defined set of equations coupling the balance equations of mass, momentum, energy and entropy with relaxation kinetics of a medium characterized by a continuous distribution of internal states is presented. We demonstrate that new phenomenological coefficients of the proposed governing equations can be extracted directly from experimental data. Our theory successfully explains the anomalous attenuation law found in experiments with biological media that is inconsistent with the conventional models using a finite number of internal degrees of freedom. The results presented offer new possibilities for medical applications of high-intensity ultrasound and ultrasound emission methods to study matter with complex internal structure. These techniques include using pressure relaxation methods for accurate investigation of fast protein folding and a variety of other applications for media where irreversible thermodynamic simulations are essential.

Simulation study of the role of the ribosomal exit tunnel on protein folding

To investigate the role of the ribosomal exit tunnel on protein folding, we simulate the initial-stage folding behavior of the protein villin headpiece subdomain HP35 (PDB id: 1yrf) with and without prefolding in the exit tunnel by using an all-atom model and find that prefolding in the exit tunnel could effectively help the protein form native secondary structures. Furthermore, our results show that, after releasing from the exit tunnel, the prefolded chains may have a tendency to form more native contacts than those only in free space and this reduces the conformational space of sampling. Our results may provide an alternative way to explain the fast folding mechanism of proteins in vivo.

Selecting fast-folding proteins by their rate of convergence

We propose a general method for predicting potentially good folders from a given number of amino acid sequences. Our approach is based on the calculation of the rate of convergence of each amino acid chain towards the native structure using only the very initial parts of the dynamical trajectories. It does not require any preliminary knowledge of the native state and can be applied to different kinds of models, including atomistic descriptions. We tested the method within both the lattice and off-lattice model frameworks and obtained several so far unknown good folders. The unbiased algorithm also allows one to determine the optimal folding temperature and takes at least 3-4 orders of magnitude fewer time steps than those needed to compute folding times.

Evolutionary Optimization of Protein Folding

Nature has shaped the make up of proteins since their appearance, 3.8 billion years ago. However, the fundamental drivers of structural change responsible for the extraordinary diversity of proteins have yet to be elucidated. Here we explore if protein evolution affects folding speed. We estimated folding times for the present-day catalog of protein domains directly from their size-modified contact order. These values were mapped onto an evolutionary timeline of domain appearance derived from a phylogenomic analysis of protein domains in 989 fully-sequenced genomes. Our results show a clear overall increase of folding speed during evolution, with known ultra-fast downhill folders appearing rather late in the timeline. Remarkably, folding optimization depends on secondary structure. While alpha-folds showed a tendency to fold faster throughout evolution, beta-folds exhibited a trend of folding time increase during the last 1.5 billion years that began during the “big bang” of domain combinations. As a consequence, these domain structures are on average slow folders today. Our results suggest that fast and efficient folding of domains shaped the universe of protein structure. This finding supports the hypothesis that optimization of the kinetic and thermodynamic accessibility of the native fold reduces protein aggregation propensities that hamper cellular functions.

Fast Folding as a Constraint in the Evolution of Protein Structures

Nature has come up with an enormous variety of protein three-dimensional structures. Structural optimization for biological function was guided by evolutionary constraints, which, however, are yet to be elucidated. Here we explore if folding speed affects protein evolution. We estimated folding times for the catalog of ∼100,000 protein domains directly from their size-modified contact order [1]. These values were mapped onto an evolutionary timeline of domain appearance derived from a phylogenomic analysis of domain abundance in 989 genomes [2]. Our results show a clear overall increase of folding speed during evolution, with known ultra-fast downhill folders appearing rather late in the timeline. Remarkably, folding optimization depends on secondary structure. While alpha-folds showed a tendency to fold faster throughout evolution, beta-folds exhibited a trend of folding time increase after the ¯¯big bang'' of domain combinations. As a consequence, these domain structures are on average slow folders today. By linking a physical measure to a phylogenomic survey, our study suggests that fast and efficient folding of domains shaped the universe of protein structure [3]. It supports the hypothesis that optimization of the kinetic accessibility of the native fold reduces detrimental protein aggregation in the cell.[1] Ivankov D-N, et al. (2003) Protein Science, 12: 2057-2062[2] Caetano-Anolles G, Caetano-Anolles D. (2003) Genome Research 13(7):1563-1571[3] Debes C, et al, in review

Fast Protein Refolding Observed in Pressure-Jump Molecular Dynamics Simulation

Pressure jump is known to induce fast protein folding. For a five-helix bundle λ-repressor fragment, a short refolding time of ∼2 μs was reported in an earlier pressure-jump experiment. To investigate this pressure-jump induced fast folding behavior, all-atom molecular dynamics simulations of more than 33 μs in explicit solvent were carried out on the same λ-repressor construct. High-pressure denatured states, generated through a high-temperature unfolding and high-pressure equilibration simulation procedure, were found to contain a significant amount of helical structure. Upon pressure drop, the protein refolded into the native state in 20 μs. The folding in the simulation is slower than the one measured in pressure-jump experiment, but faster than the folding time of 80 μs measured in temperature-jump experiment. A complete unfolding and refolding process was observed in the trajectory, which permitted the characterization of high-pressure denatured states and refolding pathway. The pressure jump simulations carried out for this study can be employed in the future to investigate slow-folding proteins through 10∼100 μs molecular dynamics simulations by inducing a fast folding phase.

Protein Folding Studied by Very Fast Pressure Drops

Temperature jump experiments have been used to study the kinetics of protein folding for over 20 years. These experiments have driven our understanding of protein folding and our attempts to simulate protein dynamics. It may be easier, however, to model and understand the effect of pressure perturbations on proteins because pressure affects biomolecular structure only through density changes, not through changes of thermal energy or solvent composition. In 2009 our lab was the first to report a pressure drop apparatus, which could be used to change the pressure of a protein solution from 2500 atm to 1 atm in less than a microsecond, causing the proteins to fold. We have recently used that instrument to study the refolding of a mutant of a protein called lambda repressor fragment 6-85 (λ∗6-85). λ∗6-85 is one of the fastest folding proteins ever discovered, with all of its known mutants folding in less than 50 μs. Surprisingly, we have observed a slow (∼1 ms) phase in the refolding of a mutant of λ∗6-85 initiated by a fast pressure drop. The emergence of this slow timescale highlights the necessity of expanding our toolbox for studying the dynamics of protein folding, as it may be that only certain conformations are populated by manipulation of a single thermodynamic variable.

Periplasmic Chaperones and their Function in the Folding of Outer Membrane Protein A into Lipid Membranes

In Gram-negative bacteria, outer membrane proteins (OMPs) are translocated across the cytoplasmic membrane in unfolded form via the Sec translocon. Prior to insertion and folding into the outer membrane, the OMPs have to traverse the periplasm. Molecular chaperones prevent OMP aggregation before the OMPs reach the outer membrane. OMPs like outer membrane protein A (OmpA) have been shown to interact with chaperones, like Skp (1-3) and SurA (4-6).OmpA folds spontaneously into lipid bilayers from a urea-unfolded form when the denaturant urea is strongly diluted. We have examined the kinetics of folding of OmpA and how it is affected by the chaperones Skp, SurA, FkpA. In all experiments, either in the absence or in the presence of any of these chaperones, the kinetics are well-described by two parallel kinetic processes of OmpA folding and insertion into lipid bilayers. Both processes were of first order. All examined chaperones increased the contribution of the faster folding process. However, only SurA caused an increase of the rate constant of the fast folding process. The temperature dependence of OmpA folding into lipid bilayers indicated that SurA lowers the activation energy of folding for the faster process.

Engineering, Characterization and usage of a Green-To-Red Photoconvertible Dronpa Mutant

The development of fluorescent proteins (FPs) and recent advances in diffraction-unlimited far-field optical microscopy have truly revolutionized our understanding of life and disease. Especially, genetic fusions of photoactivatable FPs have allowed the visualization of biological events at the nanometer scale. However, for these complicated microscopy schemes, one does not merely need a fluorescing species but rather an intelligent probe. Of remarkable interest are the photoswitchable FPs, that can be reversibly switched off and on, and the photoconvertible FPs that can be irreversibly converted from a green- to a red-emitting state. We previously reported on the rational design of a four-way optical highlighter based on Dendra2, a green-to-red photoconvertible probe (Adam et al, Chemistry & Biology, 2011). This protein, which we called NijiFP, is not only green-to-red photoconvertible, but also shows reversible photoswitching in both states. In the present work, we inverted this question and engineered a similar optical highlighter probe, this time by engineering green-to-red photoconversion properties into the photoswitchable protein Dronpa. We made ffDronpa, a Dronpa mutant that is formed up to three times as fast as Dronpa, while retaining the interesting photochromic features of Dronpa. Using rational and random mutagenesis, we transformed our fast folding Dronpa to pcDronpa. This Dronpa mutant combines Dronpa's photochromism with the feature of being photoconvertible to a red-emitting state. pcDronpa was studied in detail structurally and spectroscopically and was applied in several microscopic settings. The addition of pcDronpa to the repertoire of intelligent probes creates new opportunities for protein design and fluorescence imaging. Our example demonstrates that it is possible to elegantly introduce photodynamic behaviors in a single protein scaffold, in this case Dronpa. This work will open up new possibilities for rational and random design of FPs and their complicated photodynamic properties.

2P062 プロテインA-Bドメインの高速折り畳みダイナミクスの追跡を目指したライン共焦点顕微鏡の改良(01C.蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

2P062 プロテインA-Bドメインの高速折り畳みダイナミクスの追跡を目指したライン共焦点顕微鏡の改良(01C.蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

Effects of Macromolecular Crowding on Burst Phase Kinetics of Cytochrome c Folding

Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.

A Buried Ionizable Residue Destabilizes the Native State and the Transition State in the Folding of Monellin

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.

Fast Photochemical Oxidation of Proteins and Mass Spectrometry Follow Submillisecond Protein Folding at the Amino-Acid Level

We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast photochemical oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose "protection" changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix(1) shows a decreasing trend of oxidative modification during the first 0.1-1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix(1) formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the submillisecond time range.

Protein Folding: Adding a Nucleus to Guide Helix Docking Reduces Landscape Roughness

The elongated three-helix‐bundle spectrin domains R16 and R17 fold and unfold unusually slowly over a rough energy landscape, in contrast to the homologue R15, which folds fast over a much smoother, more typical landscape. R15 folds via a nucleation–condensation mechanism that guides the docking of the A and C-helices. However, in R16 and R17, the secondary structure forms first and the two helices must then dock in the correct register. Here, we use variants of R16 and R17 to demonstrate that substitution of just five key residues is sufficient to alter the folding mechanism and reduce the landscape roughness. We suggest that, by providing access to an alternative, faster, folding route over their landscape, R16 and R17 can circumvent their slow, frustrated wild-type folding mechanism.

Fast Subdomain Folding Prior to the Global Refolding Transition of E. coli Adenylate Kinase: A Double Kinetics Study

The rate of folding of globular proteins depends on specific local and nonlocal intramolecular interactions. What is the relative role of these two types of interaction at the initiation of refolding? We address this question by application of a "double kinetics" method based on fast initiation of refolding of site specifically labeled protein samples and detection of the transient distributions of selected intramolecular distances by means of fast measurements of time-resolved fluorescence resonance energy transfer. We determined the distribution of the distance between the ends of a 44-chain segment that includes the AMP(bind) domain, by labeling residues 28 and 71, in Escherichia coli adenylate kinase (AK) and the distribution of the distance between residues 18 and 203, which depends on the overall order of the molecule. That distribution shows two-state transition to the native intramolecular distance at the same rate as that of the cooperative refolding transition of the AK molecule. In sharp contrast, the distance distribution between residues 28 and 71 is already native like at the end of the dead-time of the mixing device. This fast formation of native short distance between two widely separated chain sections can be either dependent on fast folding of the AMP(bind) domain or a result of a very effective nonlocal interaction between specific short clusters of hydrophobic residues. Further experiments on studying the kinetics of folding of selected structural elements in the protein will help determination of the driving force of this early folding event.

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state.

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R 2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.

Protein folding kinetics and thermodynamics from atomistic simulation

Advances in simulation techniques and computing hardware have created a substantial overlap between the timescales accessible to atomic-level simulations and those on which the fastest-folding proteins fold. Here we demonstrate, using simulations of four variants of the human villin headpiece, how simulations of spontaneous folding and unfolding can provide direct access to thermodynamic and kinetic quantities such as folding rates, free energies, folding enthalpies, heat capacities, Φ-values, and temperature-jump relaxation profiles. The quantitative comparison of simulation results with various forms of experimental data probing different aspects of the folding process can facilitate robust assessment of the accuracy of the calculations while providing a detailed structural interpretation for the experimental observations. In the example studied here, the analysis of folding rates, Φ-values, and folding pathways provides support for the notion that a norleucine double mutant of villin folds five times faster than the wild-type sequence, but following a slightly different pathway. This work showcases how computer simulation has now developed into a mature tool for the quantitative computational study of protein folding and dynamics that can provide a valuable complement to experimental techniques.