Downhill Folding Research Articles

Self-assembly of emulsion droplets through programmable folding.

In the realm of particle self-assembly, it is possible to reliably construct nearly arbitrary structures if all the pieces are distinct1-3, but systems with fewer flavours of building blocks have so far been limited to the assembly of exotic crystals4-6. Here we introduce a minimal model system of colloidal droplet chains7, with programmable DNA interactions that guide their downhill folding into specific geometries. Droplets are observed in real space and time, unravelling the rules of folding. Combining experiments, simulations and theory, we show that controlling the order in which interactions are switched on directs folding into unique structures, which we call colloidal foldamers8. The simplest alternating sequences (ABAB...) of up to 13 droplets yield 11 foldamers in two dimensions and one in three dimensions. Optimizing the droplet sequence and adding an extra flavour uniquely encodes more than half of the 619 possible two-dimensional geometries. Foldamers consisting of at least 13 droplets exhibit open structures with holes, offering porous design. Numerical simulations show that foldamers can further interact to make complex supracolloidal architectures, such as dimers, ribbons and mosaics. Our results are independent of the dynamics and therefore apply to polymeric materials with hierarchical interactions on all length scales, from organic molecules all the way to Rubik's Snakes. This toolbox enables the encoding of large-scale design into sequences of short polymers, placing folding at the forefront of materials self-assembly.

Magnetic tweezers to observe the ultrafast downhill folding of a protein in the millisecond and nanometer scale

Recent advances in magnetic tweezers set them as a promising tool to unveil the dynamics of proteins and nucleic acids by means of hours to days-long stable constant force experiments. Archetypal folds play a crucial role in our means to uncover the intricacies of protein folding. One prominent case is the λ-phage cI transcription repressor fragment λ6-85; a five-helix bundle protein were single point mutations shift its folding time from fast 200 μs to ultrafast downhill-like 2.3 μs kinetics. Can we resolve ultrafast kinetics with the milliseconds time-resolution of the magnetic tweezers? Here, we report the observation of ultrafast protein folding in magnetic tweezers.

NMR Analysis of Protein Folding Interaction Networks.

Theory and experimental evidence unequivocally indicate that protein folding is far more complex than the two-state (all-or-none) model that is usually assumed in the analysis of folding experiments. Proteins tend to fold hierarchically by forming secondary structure elements, followed by supersecondary arrangements, and other intermediate states that ultimately adopt the native tertiary fold as a result of a delicate balance between interatomic interactions and entropic contributions. However, small proteins with simple folds typically follow downhill folding, characterized by very small energetic barriers (<3RT) that allow multiple protein conformations to be populated along the folding path down the free energy landscape, reaching the native fold at the lowest energy level.Here we describe the use of solution-state nuclear magnetic resonance (NMR) for the analysis of protein folding interaction networks at atomic resolution. The assignment of NMR spectra acquired at different unfolding conditions provides hundreds of atomic unfolding curves that are analyzed to infer the network of folding interactions. The method is particularly useful to study small proteins that fold autonomously in the sub-millisecond timescale. The information obtained from the application of this method can potentially unveil the basic relationships between protein structure and folding.

The change of conditions does not affect Ros87 downhill folding mechanism

Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability. Downhill folders are estimated to be rather rare in nature as they miss an energetically substantial folding barrier that can protect against aggregation and proteolysis. We have previously demonstrated that the prokaryotic zinc finger protein Ros87 shows a bipartite folding/unfolding process in which a metal binding intermediate converts to the native structure through a delicate barrier-less downhill transition. Significant variation in folding scenarios can be detected within protein families with high sequence identity and very similar folds and for the same sequence by varying conditions. For this reason, we here show, by means of DSC, CD and NMR, that also in different pH and ionic strength conditions Ros87 retains its partly downhill folding scenario demonstrating that, at least in metallo-proteins, the downhill mechanism can be found under a much wider range of conditions and coupled to other different transitions. We also show that mutations of Ros87 zinc coordination sphere produces a different folding scenario demonstrating that the organization of the metal ion core is determinant in the folding process of this family of proteins.

Substitution of the Native Zn(II) with Cd(II), Co(II) and Ni(II) Changes the Downhill Unfolding Mechanism of Ros87 to a Completely Different Scenario.

The structural effects of zinc replacement by xenobiotic metal ions have been widely studied in several eukaryotic and prokaryotic zinc-finger-containing proteins. The prokaryotic zinc finger, that presents a bigger βββαα domain with a larger hydrophobic core with respect to its eukaryotic counterpart, represents a valuable model protein to study metal ion interaction with metallo-proteins. Several studies have been conducted on Ros87, the DNA binding domain of the prokaryotic zinc finger Ros, and have demonstrated that the domain appears to structurally tolerate Ni(II), albeit with important structural perturbations, but not Pb(II) and Hg(II), and it is in vitro functional when the zinc ion is replaced by Cd(II). We have previously shown that Ros87 unfolding is a two-step process in which a zinc binding intermediate converts to the native structure thorough a delicate downhill folding transition. Here, we explore the folding/unfolding behaviour of Ros87 coordinated to Co(II), Ni(II) or Cd(II), by UV-Vis, CD, DSC and NMR techniques. Interestingly, we show how the substitution of the native metal ion results in complete different folding scenarios. We found a two-state unfolding mechanism for Cd-Ros87 whose metal affinity Kd is comparable to the one obtained for the native Zn-Ros87, and a more complex mechanism for Co-Ros87 and Ni-Ros87, that show higher Kd values. Our data outline the complex cross-correlation between the protein–metal ion equilibrium and the folding mechanism proposing such an interplay as a key factor in the proper metal ion selection by a specific metallo-protein.

Lessons about Protein Folding and Binding from Archetypal Folds.

The function of proteins as biological nanomachines relies on their ability to fold into complex 3D structures, bind selectively to partners, and undergo conformational changes on cue. The native functional structures, and the rates of interconversion between conformational states (folded-unfolded, bound-free), are all encoded in the physical chemistry of their amino acid sequence. However, despite extensive research over decades, this code has proven difficult to fully crack, in terms of both prediction and understanding the molecular mechanisms at play.Earlier work on single-domain proteins reported a commonality of slow rates (10-2-102 s-1) and simple behavior in both kinetic and thermodynamic unfolding experiments, which suggested the process was all-or-none and thereby analogous to a chemical reaction (e.g., A ⇄ B). In the absence of a first-principles pre-exponential factor for protein (un)folding dynamics, the rates could only be interpreted in relative terms, e.g., the changes induced by mutation, and hence, neither the height of nor the entropic contribution to the free energy barriers was known. The rates were also many orders of magnitude too slow for direct atomistic simulations, and the computational focus was on predicting rate changes induced by mutation via coarse grained simulations. However, even the effects of mutation proved to be strikingly homogeneous with all experimental data clustering at ∼1/3 of the free energy perturbation recovered on folding and ∼2/3 on unfolding.The implementation of ultrafast kinetic methods turned the field upside down because they allowed researchers to measure the time scales of elementary (un)folding motions, which set the pre-exponential factor for protein conformational transitions at ∼1 μs. In parallel, we and others set out to investigate the simplest possible protein structures capable of autonomous folding, which we defined as archetypal folds. The rationale was to recapitulate the hierarchical organization of protein structure, starting from the bottom up. The study of fold archetypes ended up opening new research avenues in protein (un)folding, but also making unexpected connections with the folding upon binding of intrinsically disordered proteins and suggesting their functioning as conformational rheostats.This Account describes our work on the kinetic, thermodynamic, mechanistic, and functional analysis of fold archetypes. We first discuss the kinetic studies, emphasizing their impact on our understanding of (un)folding rates, of barrierless (downhill) folding, and as benchmarks for atomistic simulations. We continue with the thermodynamic analysis, introducing the differential scanning calorimetry, multiprobe, and NMR approaches that we developed to dissect their gradual, minimally cooperative (un)folding transitions and to probe the underlying mechanisms with unprecedented detail. The last two sections cover single-molecule analyses and some recent, mostly computational, results on the exploration of possible biological and technological roles for the gradual conformational transitions of fold archetypes.

Downhill (Un)Folding Coupled to Binding as a Mechanism for Engineering Broadband Protein Conformational Transducers.

Canonical proteins fold and function as conformational switches that toggle between their folded (on) and unfolded (off) states, a mechanism that also provides the basis for engineering transducers for biosensor applications. One of the limitations of such transducers, however, is their relatively narrow operational range, limited to ligand concentrations 20-fold below or above their C50. Previously, we discovered that certain fast-folding proteins lose/gain structure gradually (downhill folding), which led us to postulate their operation as conformational rheostats capable of processing inputs/outputs in analog fashion. Conformational rheostats could make transducers with extended sensitivity. Here we investigate this hypothesis by engineering pH transducing into the naturally pH insensitive, downhill folding protein gpW. Particularly, we engineered histidine grafts into its hydrophobic core to induce unfolding via histidine ionization. We designed and tested the effects of ionization via computational modeling and studied experimentally the four most promising single grafts and two double grafts. All tested mutants become reversible pH transducers in the 4-9 range, and their response increases proportionally to how buried the histidine graft is. Importantly, the pH-dependent reversible (un)folding occurs in rheostatic fashion, so the engineered transducers can detect up to 6 orders of magnitude in [H+] for single grafts, and even more for double grafts. Our results demonstrate that downhill (un)folding coupled to binding produces the gradual, analog responses to the ligand (here H+) that are expected of conformational rheostats, and which make them a powerful mechanism for engineering transducers with sensitivity over many orders of magnitude in ligand concentration (broadband).

A new way to recognize downhill folding based on generalized path length

The protein folding is an important scientific problem and many methods were designed to elucidate the protein folding and obtain insight into the molecular mechanism. A novel means is presented to identify the downhill pathways of protein folding in this paper. This method is based on barrier energy profile projected onto the generalized path length (GPL) with Breadth-first searching (BFS) algorithm. We show the effectiveness of this approach by constructing the barrier energy profile of trpzip2 and comparing with other methods.

Different potential of mean force of two-state protein GB1 and downhill protein gpW revealed by molecular dynamics simulation**Project supported by the National Natural Science Foundation of China (Grant Nos. 11874309, 11474237, and 11574310) and the 111 Project, China (Grant No. B16029).

Two-state folding and down-hill folding are two kinds of protein folding dynamics for small single domain proteins. Here we apply molecular dynamics (MD) simulation to the two-state protein GB1 and down-hill folding protein gpW to reveal the relationship of their free energy landscape and folding/unfolding dynamics. Results from the steered MD simulations show that gpW is much less mechanical resistant than GB1, and the unfolding process of gpW has more variability than that of GB1 according to their force–extension curves. The potential of mean force (PMF) of GB1 and gpW obtained by the umbrella sampling simulations shows apparent difference: PMF of GB1 along the coordinate of extension exhibits a kink transition point where the slope of PMF drops suddenly, while PMF of gpW increases with extension smoothly, which are consistent with two-state folding dynamics of GB1 and downhill folding dynamics of gpW, respectively. Our results provide insight to understand the fundamental mechanism of different folding dynamics of two-state proteins and downhill folding proteins.

New Insights into Folding, Misfolding, and Nonfolding Dynamics of a WW Domain.

Intermediate states in protein folding are associated with formation of amyloid fibrils, which are responsible for a number of neurodegenerative diseases. Therefore, prevention of the aggregation of folding intermediates is one of the most important problems to overcome. Recently, we studied the origins and prevention of formation of intermediate states with the example of the Formin binding protein 28 (FBP28) WW domain. We demonstrated that the replacement of Leu26 by Asp26 or Trp26 (in ∼15% of the folding trajectories) can alter the folding scenario from three-state folding, a major folding scenario for the FBP28 WW domain (WT) and its mutants, toward two-state or downhill folding at temperatures below the melting point. Here, for a better understanding of the physics of the formation/elimination of intermediates, (i) the dynamics and energetics of formation of β-strands in folding, misfolding, and nonfolding trajectories of these mutants (L26D and L26W) is investigated; (ii) the experimental structures of WT, L26D, and L26W are analyzed in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger equation. We show that the formation of each β-strand in folding trajectories is accompanied by the emergence of kinks in internal coordinate space as well as a decrease in local free energy. In particular, the decrease in downhill folding trajectory is ∼7 kcal/mol, while it varies between 31 and 48 kcal/mol for the three-state folding trajectory. The kink analyses of the experimental structures give new insights into formation of intermediates, which may become a useful tool for preventing aggregation.

In-Cell Protein–Protein Contacts: Transient Interactions in the Crowd

Proteins in vivo are immersed in a crowded environment of water, ions, metabolites, and macromolecules. In-cell experiments highlight how transient weak protein-protein interactions promote (via functional "quinary structure") or hinder (via competitive binding or "sticking") complex formation. Computational models of the cytoplasm are expensive. We tackle this challenge with an all-atom model of a small volume of the E. coli cytoplasm to simulate protein-protein contacts up to the 5 μs time scale on the special-purpose supercomputer Anton 2. We use three CHARMM-derived force fields: C22*, C36m, and C36mCU (with CUFIX corrections). We find that both C36m and C36mCU form smaller contact surfaces than C22*. Although CUFIX was developed to reduce protein-protein sticking, larger contacts are observed with C36mCU than C36m. We show that the lifespan Δt of protein-protein contacts obeys a power law distribution between 0.03 and 3 μs, with ∼90% of all contacts lasting <1 μs (similar to the time scale for downhill folding).

Instrumental Effects in the Dynamics of an Ultrafast Folding Protein under Mechanical Force.

The analysis and interpretation of single molecule force spectroscopy (smFS) experiments is often complicated by hidden effects from the measuring device. Here we investigate these effects in our recent smFS experiments on the ultrafast folding protein gpW, which has been previously shown to fold without crossing a free energy barrier in the absence of force (i.e., downhill folding). Using atomic force microscopy (AFM) smFS experiments, we found that a very small force of ∼5 pN brings gpW near its unfolding midpoint and results in two-state (un)folding patterns that indicate the emergence of a force-induced free energy barrier. The change in the folding regime is concomitant with a 30,000-fold slowdown of the folding and unfolding times, from a few microseconds that it takes gpW to (un)fold at the midpoint temperature to seconds in the AFM. These results are puzzling because the barrier induced by force in the folding free energy landscape of gpW is far too small to account for such a difference in time scales. Here we use recently developed theoretical methods to resolve the origin of the strikingly slow dynamics of gpW under mechanical force. We find that, while the AFM experiments correctly capture the equilibrium distance distribution, the measured dynamics are entirely controlled by the response of the cantilever and polyprotein linker, which is much slower than the protein conformational dynamics. This interpretation is likely applicable to the folding of other small biomolecules in smFS experiments, and becomes particularly important in the case of systems with fast folding dynamics and small free energy barriers, and for instruments with slow response times.

Protein Folding Cooperativity and Thermodynamic Barriers of the Simplest β-Sheet Fold: A Survey of WW Domains.

Theory and experiments have shown that microsecond folding proteins exhibit characteristic thermodynamic properties that reflect the limited cooperativity of folding over marginal barriers (downhill folding). Those studies have mostly focused on proteins with large α-helical contents and small size, which tend to be the fastest folders. A key open question is whether such properties are also present in the fastest all-β proteins. We address this issue by investigating the unfolding thermodynamics of a collection of WW domains as representatives of the simplest β-sheet fold. WW domains are small microsecond folders, although they do not fold as fast as their α-helical counterparts. In previous work on the NEDD4-WW4 domain, we reported deviations from two-state thermodynamics that were less apparent and thus suggestive of an incipient downhill scenario. Here we investigate the unfolding thermodynamics of four other WW domains (NEDD4-WW3, YAP65-WW1(L30K), FBP11-WW1, and FBP11-WW2) by performing all of the thermodynamic tests for downhill folding that have been previously developed on α-helical proteins. This set of five WW domains shares low sequence identity and include examples from two specificity classes, thus providing a comprehensive survey. Thermodynamic analysis of the four new WW domains consistently reveals all of the properties of downhill folding equilibria, which are in all cases more marked than what we found before in NEDD4-WW4. Our results show that fast-folding all-β proteins do share limited cooperativity and gradual unfolding thermodynamics with fast α-helical proteins and suggest that the free energy barrier to folding of natural proteins is mostly determined by size and fold topology and much less by the specific amino acid sequence.

A non-equilibrium approach to allosteric communication.

While the theory of protein folding is well developed, including concepts such as rugged energy landscape, folding funnel, etc., the same degree of understanding has not been reached for the description of the dynamics of allosteric transitions in proteins. This is not only due to the small size of the structural change upon ligand binding to an allosteric site, but also due to challenges in designing experiments that directly observe such an allosteric transition. On the basis of recent pump-probe-type experiments (Buchli et al. 2013 Proc. Natl Acad. Sci. USA110, 11 725-11 730. (doi:10.1073/pnas.1306323110)) and non-equilibrium molecular dynamics simulations (Buchenberg et al. 2017 Proc. Natl Acad. Sci. USA114, E6804-E6811. (doi:10.1073/pnas.1707694114)) studying an photoswitchable PDZ2 domain as model for an allosteric transition, we outline in this perspective how such a description of allosteric communication might look. That is, calculating the dynamical content of both experiment and simulation (which agree remarkably well with each other), we find that allosteric communication shares some properties with downhill folding, except that it is an 'order-order' transition. Discussing the multiscale and hierarchical features of the dynamics, the validity of linear response theory as well as the meaning of 'allosteric pathways', we conclude that non-equilibrium experiments and simulations are a promising way to study dynamical aspects of allostery.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Allostery and molecular machines.

Machines undergo repeated energy (fuel)-driven cycling between various functional and structural states. Such cycling involves motions of the machine parts, which take place in a highly coordinated manner in time and space. This description applies not only to man-made machines but also to the molecular machines that mediate many of the key processes in all forms of life [1]. Examples include protein synthesis by the ribosome, DNA unwinding by helicases during replication, protein degradation by the proteasome and protein folding by chaperones. The coordinated movements of most biomolecular machines are achieved by allosteric regulation of ATP binding and hydrolysis. Consequently, a molecular level understanding of how the essential machines of life work requires invoking and further developing allosteric theory. Hence, the motivation for the Royal Society Discussion Meeting entitled ‘Allostery and molecular machines' that took place in June 2017. The meeting brought together scientists interested in allostery, in general, with others who are studying specific biomolecular machines of interest. The meeting led to many interesting discussions and resulted in this special issue of the Philosophical Transactions B . In 1965, Monod, Wyman & Changeux published a seminal paper [2] that described a so-called ‘concerted model' (referred to here and generally as the MWC model) for cooperative ligand binding by oligomeric proteins. This issue contains a paper by one of the co-authors of this landmark paper in which the MWC model is explained in brief and its application to the nicotinic acetylcholine receptor is then described [3]. The MWC model remains important but its elegance has come at the price of several restrictive assumptions. One key assumption is that cooperativity is due to a shift in equilibrium between different quaternary …

Tertiary Interactions in the Unbound Guanine-Sensing Riboswitch Focus Functional Conformational Variability on the Binding Site

Riboswitches are genetic regulatory elements mainly found in bacteria, which regulate gene expression based on the availability of a ligand. Purine-sensing riboswitches, including the guanine-sensing riboswitch (Gsw), possess tertiary interactions connecting the L2 and L3 loops. These interactions are important for ligand binding to the aptamer. However, atomic-level structural knowledge about the unbound state and how the tertiary interactions influence the conformational heterogeneity of the aptamer is still scarce. We performed replica exchange molecular dynamics simulations of the aptamer domain of wild-type Gsw and a G37A/C61U mutant, which exhibits destabilized tertiary interactions, at different Mg2+ concentrations with an aggregate simulation time of ∼16 μs, and subsequently obtained free-energy landscapes. Our data provide evidence that suggests that the unbound state of wild-type Gsw is conformationally rather homogeneous from a global viewpoint, yet the ligand binding site shows functionally necessary mobility required for ligand binding. For the mutant, the data suggest a heterogeneous ensemble, in particular without Mg2+. Hence, the tertiary interactions focus functional conformational variability on the binding site region of wild-type Gsw. Our data allow speculating that already the weakening of the tertiary interactions by two hydrogen bonds shifts the kinetics of folding from downhill folding without traps or intermediate states for wild-type Gsw to a folding including intermediates and misfolded structures for the mutant. A slowed-down folding of the aptamer might favor a decision during transcriptional regulation for the off-path, even if the ligand binds.

Roles of conformational disorder and downhill folding in modulating protein-DNA recognition.

Transcription factors are thought to efficiently search for their target DNA site via a combination of conventional 3D diffusion and 1D diffusion along the DNA molecule mediated by non-specific electrostatic interactions. This process requires the DNA-binding protein to quickly exchange between a search competent and a target recognition mode, but little is known as to how these two binding modes are encoded in the conformational properties of the protein. Here, we investigate this issue on the engrailed homeodomain (EngHD), a DNA-binding domain that folds ultrafast and exhibits a complex conformational behavior consistent with the downhill folding scenario. We explore the interplay between folding and DNA recognition using a coarse-grained computational model that allows us to manipulate the folding properties of the protein and monitor its non-specific and specific binding to DNA. We find that conformational disorder increases the search efficiency of EngHD by promoting a fast gliding search mode in addition to sliding. When gliding, EngHD remains loosely bound to DNA moving linearly along its length. A partially disordered EngHD also binds more dynamically to the target site, reducing the half-life of the specific complex via a spring-loaded mechanism. These findings apply to all conditions leading to partial disorder. However, we also find that at physiologically relevant temperatures EngHD is well folded and can only obtain the conformational flexibility required to accelerate 1D diffusion when it folds/unfolds within the downhill scenario (crossing a marginal free energy barrier). In addition, the conformational flexibility of native downhill EngHD enables its fast reconfiguration to lock into the specific binding site upon arrival, thereby affording finer control of the on- and off-rates of the specific complex. Our results provide key mechanistic insights into how DNA-binding domains optimize specific DNA recognition through the control of their conformational dynamics and folding mechanism.

Eliminating a Protein Folding Intermediate by Tuning a Local Hydrophobic Contact.

Intermediate states in protein folding may slow folding, and sometimes can provide a starting point for aggregation. Recently, the FBP28 WW domain of the formin-binding protein was used as a model for a computational study of the origin and prevention of intermediate-state formation, and local hydrophobic interactions of Leu26 were implicated. Here, we combine new simulations over a broad temperature range with experimental temperature-jump data to study this site in more detail. We replace Leu26 by Asp26 or Trp26 to alter the folding scenario from three-state folding toward two-state or downhill folding at temperatures below the melting point, whereas the wild type shows two-state behavior only near its melting temperature. We offer an explanation of this behavior mainly in terms of principles of hydrophobic interactions.

When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.

Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico. All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats.

Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange

The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λYA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10(5) s(-1) and a stability of 7.4 kcal·mol(-1) at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions.