Refolding Events Research Articles

Complexity of Guanine Quadruplex Unfolding Pathways Revealed by Atomistic Pulling Simulations.

Guanine quadruplexes (GQs) are non-canonical nucleic acid structures involved in many biological processes. GQs formed in single-stranded regions often need to be unwound by cellular machinery, so their mechanochemical properties are important. Here, we performed steered molecular dynamics simulations of human telomeric GQs to study their unfolding. We examined four pulling regimes, including a very slow setup with pulling velocity and force load accessible to high-speed atomic force microscopy. We identified multiple factors affecting the unfolding mechanism, i.e.,: (i) the more the direction of force was perpendicular to the GQ channel axis (determined by GQ topology), the more the base unzipping mechanism happened, (ii) the more parallel the direction of force was, GQ opening and cross-like GQs were more likely to occur, (iii) strand slippage mechanism was possible for GQs with an all-anti pattern in a strand, and (iv) slower pulling velocity led to richer structural dynamics with sampling of more intermediates and partial refolding events. We also identified that a GQ may eventually unfold after a force drop under forces smaller than those that the GQ withstood before the drop. Finally, we found out that different unfolding intermediates could have very similar chain end-to-end distances, which reveals some limitations of structural interpretations of single-molecule spectroscopic data.

Structure and epitope of a neutralizing monoclonal antibody that targets the stem helix of β coronaviruses.

Monoclonal antibodies that retain neutralizing activity against multiple coronavirus (CoV) lineages and variants of concern (VoC) must be developed to protect against future pandemics. These broadly neutralizing MAbs (BNMAbs) may be used as therapeutics and/or to assist in the rational design of vaccines that induce BNMAbs. 1249A8 is a BNMAb that targets the stem helix (SH) region of CoV spike (S) protein and neutralizes Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) original strain, delta, and omicron VoC, Severe Acute Respiratory Syndrome CoV (SARS-CoV), and Middle East Respiratory Syndrome CoV (MERS-CoV). To understand its mechanism of action, the crystal structure of 1249A8 bound to a MERS-CoV SH peptide was determined at 2.1 Å resolution. BNMAb 1249A8 mimics the SARS-CoV-2 S loop residues 743-749, which interacts with the N-terminal end of the SH helix in the S post-fusion conformation. The conformation of 1249A8-bound SH is distinct from the SH conformation observed in the post-fusion SARS-CoV-2 S structure, suggesting 1249A8 disrupts the secondary structure and refolding events required for CoV post-fusion S to initiate membrane fusion and ultimately infection. This study provides novel insights into the neutralization mechanisms of SH-targeting CoV BNMAbs that may inform vaccine development and the design of optimal BNMAb therapeutics.

RNA modifications in structure prediction - Status quo and future challenges.

Chemical modifications of RNA nucleotides change their identity and characteristics and thus alter genetic and structural information encoded in the genomic DNA. tRNA and rRNA are probably the most heavily modified genes, and often depend on derivatization or isomerization of their nucleobases in order to correctly fold into their functional structures. Recent RNomics studies, however, report transcriptome wide RNA modification and suggest a more general regulation of structuredness of RNAs by this so called epitranscriptome. Modification seems to require specific substrate structures, which in turn are stabilized or destabilized and thus promote or inhibit refolding events of regulatory RNA structures. In this review, we revisit RNA modifications and the related structures from a computational point of view. We discuss known substrate structures, their properties such as sub-motifs as well as consequences of modifications on base pairing patterns and possible refolding events. Given that efficient RNA structure prediction methods for canonical base pairs have been established several decades ago, we review to what extend these methods allow the inclusion of modified nucleotides to model and study epitranscriptomic effects on RNA structures.

Directly monitor protein rearrangement on a nanosecond-to-millisecond time-scale

In order to directly observe the refolding kinetics from a partially misfolded state to a native state in the bottom of the protein-folding funnel, we used a “caging” strategy to trap the β-sheet structure of ubiquitin in a misfolded conformation. We used molecular dynamics simulation to generate the cage-induced, misfolded structure and compared the structure of the misfolded ubiquitin with native ubiquitin. Using laser flash irradiation, the cage can be cleaved from the misfolded structure within one nanosecond, and we monitored the refolding kinetics of ubiquitin from this misfolded state to the native state by photoacoustic calorimetry and photothermal beam deflection techniques on nanosecond to millisecond timescales. Our results showed two refolding events in this refolding process. The fast event is shorter than 20 ns and corresponds to the instant collapse of ubiquitin upon cage release initiated by laser irradiation. The slow event is ~60 μs, derived from a structural rearrangement in β-sheet refolding. The event lasts 10 times longer than the timescale of β-hairpin formation for short peptides as monitored by temperature jump, suggesting that rearrangement of a β-sheet structure from a misfolded state to its native state requires more time than ab initio folding of a β-sheet.

Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion.

The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.

Benchmarking all-atom simulations using hydrogen exchange.

Long-time molecular dynamics (MD) simulations are now able to fold small proteins reversibly to their native structures [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520]. These results indicate that modern force fields can reproduce the energy surface near the native structure. To test how well the force fields recapitulate the other regions of the energy surface, MD trajectories for a variant of protein G are compared with data from site-resolved hydrogen exchange (HX) and other biophysical measurements. Because HX monitors the breaking of individual H-bonds, this experimental technique identifies the stability and H-bond content of excited states, thus enabling quantitative comparison with the simulations. Contrary to experimental findings of a cooperative, all-or-none unfolding process, the simulated denatured state ensemble, on average, is highly collapsed with some transient or persistent native 2° structure. The MD trajectories of this protein G variant and other small proteins exhibit excessive intramolecular H-bonding even for the most expanded conformations, suggesting that the force fields require improvements in describing H-bonding and backbone hydration. Moreover, these comparisons provide a general protocol for validating the ability of simulations to accurately capture rare structural fluctuations.

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting.

To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.

Fusion activation by a headless parainfluenza virus 5 hemagglutinin-neuraminidase stalk suggests a modular mechanism for triggering

The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion.

Touring the Protein Energy Landscape: The View Depends on Where and How You Look

Decoding the information in the primary sequence of a protein is one of the most fundamental challenges in modern biology. A protein's sequence encodes more than just the native structure; it encodes the entire energy landscape – an ensemble of conformations whose energetics and dynamics are finely tuned. I will present developments in both our single molecule and ensemble studies probing high‐energy fluctuations on the energy landscape. In particular, we have use a novel method to explore the mechanical stability of single protein molecules in the low force regime of the laser tweezers. The low spring constant of the laser tweezers enables us to directly monitor low force refolding events and fluctuations between different molecular structures at quasi‐equilibrium conditions. Using this approach we have been able to identify and monitor novel events on the conformational energy landscape. Our recent results on the differences in mechanical compliance between the molten globule and native states and force‐dependent changes in the unfolding pathway of proteins have important implications for mechanical processes in the cell. I will also present our development of a thiol‐exchange method that allows partitioning of fluctuations and partially unfolded conformations to either side of the rate limiting step for unfolding.

Tertiary DNA Structure in the Single-Stranded hTERT Promoter Fragment Unfolds and Refolds by Parallel Pathways via Cooperative or Sequential Events

The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but also this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells.

ChemInform Abstract: The Interplay of Turn Formation and Hydrophobic Interactions on the Early Kinetic Events in Protein Folding

AbstractReview: 67 refs.

The interplay of turn formation and hydrophobic interactions on the early kinetic events in protein folding

While both turn formation and hydrophobic interactions play dominant roles in the initiation of protein folding, their individual contributions to the folding kinetics and to the structural stability of the protein still remain poorly understood. Here, we applied a photolabile linker to "cage" some important structural motifs, including both α-helices and β-sheets, into their non-native states. These "caged" structural motifs are then relaxed by laser-flash photolysis and their refolding events followed by photoacoustic calorimetry (PAC) and photothermal beam deflection (PBD). These experiments, combined with our previous results, revealed that spontaneous α-helix formation can occur extremely rapidly (10(8)-10(9) s(-1)) if the process is driven solely by turn formation followed by helix propagation. However, if sequestering of the side chains of hydrophobic amino acid residues participates in the refolding process, which may provide additional driving force beyond that afforded by turn formation alone, the refolding rate will be retarded, often by many orders of magnitude. This is usually the case in the formation of three-stranded β-sheets (10(7)-10(8) s(-1)) and β-hairpins (10(5)-10(6) s(-1)). Thus, we propose that proteins take advantage of the hierarchy of timescales associated with either turn formation, hydrophobic interactions, or global collapse of tertiary structure to accomplish the folding process in an orderly fashion, as these events are sufficiently separated in time and do not interfere with one another.

Single-Molecule Protein Unfolding and Translocation by the AAA+ Protease, ClpXP

All cells employ ATP-fueled AAA+ proteases for protein-quality control and regulation. In the ClpXP protease, the ring-shaped AAA+ molecular motor ClpX first recognizes and mechanically unfolds specific protein substrates, then translocates the denatured polypeptide through a central axial pore into the barrel-shaped peptidase ClpP for degradation. Although the mechanisms of ATP utilization and substrate degradation have been extensively studied in solution and recently at the single-molecule level[1, 2], the molecular details underlying mechanical substrate unfolding and stepping along the polypeptide track remain unexamined.Using a dual-laser optical trap, we probed how ClpXP unfolds and translocates a multi-domain protein substrate at the single-molecule level. In our assay, a ClpXP-substrate complex is tethered between two trapped polystyrene beads held in a dumbbell configuration [2]. Motility records of ClpXP along the polypeptide track show unique, fingerprint-like, substrate unfolding and translocation events. Following rapid and cooperative unfolding of individual domains, we find that ClpX translocates the polypeptide into ClpP, taking small steps of 5-8 amino acids. The nature of the polypeptide track affects ClpXP mechanochemical activity. ClpX step size does not depend on ClpP, though we observe substantial substrate refolding and slippage events when only ClpX is examined. Our results support a power-stroke model of denaturation in which successful unfolding requires mechanical pulling by the enzyme to coincide with transient stochastic protein destabilization.[1] Shin, Y., Davis, J.H., Brau, R.R., Martin, A., Kenniston, J.A., Baker T.A., Sauer, R.T., Lang, M.J. Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease. PNAS, 106, 19340 (2009)[2] Aubin-Tam, M. E., Olivares, A. O., Sauer, R. T., Baker, T. A., Lang, M. J. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell, 145, 257-67 (2011)

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use (15)N, , and (13)CO NMR R(2) relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R(2) dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75-4.95 and determined to be an on-pathway intermediate (I1) in a linear three-state unfolding scheme (N&lrarr2;I1&lrarr2;MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N → I1 (36 s(-1)) and MG → I1 (26 s(-1)), respectively. Differences in chemical shift between N and I1 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R(2) dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N → MG transition. The experiments also identify regions of energetic frustration that "crack" during unfolding and impede the refolding process.

Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and is [corrected] under severe conformational constraints [corrected] dictated by the one-dimensional geometry of the tunnel. [corrected] How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.

Multiple Unfolding Events during Native Folding of the Tetrahymena Group I Ribozyme

Despite the ubiquitous nature of misfolded intermediates in RNA folding, little is known about their physical properties or the folding transitions that allow them to continue folding productively. Folding of the Tetrahymena group I ribozyme includes sequential accumulation of two intermediates, termed I trap and misfolded (M). Here, we probe the structure and folding transition of I trap and compare them to those of M. Hydroxyl radical and dimethyl sulfate footprinting show that both I trap and M are extensively structured and crudely resemble the native RNA. However, regions of the core P3–P8 domain are more exposed to solvent in I trap than in M. I trap rearranges to continue folding nearly 1000-fold faster than M, and urea accelerates folding of I trap much less than M. Thus, the rate-limiting transition from I trap requires a smaller increase in exposed surface. Mutations that disrupt peripheral tertiary contacts give large and nearly uniform increases in re-folding of M, whereas the same mutations give at most modest increases in folding from I trap. Intriguingly, mutations within the peripheral element P5abc give 5- to 10-fold accelerations in escape from I trap, whereas ablation of P13, which lies on the opposite surface in the native structure, near the P3–P8 domain, has no effect. Thus, the unfolding required from I trap appears to be local, whereas the unfolding of M appears to be global. Further, the modest effects from several mutations suggest that there are multiple pathways for escape from I trap and that escape is aided by loosening nearby native structural constraints, presumably to facilitate local movements of nucleotides or segments that have not formed native contacts. Overall, these and prior results suggest a model in which the global architecture and peripheral interactions of the RNA are achieved relatively early in folding. Multiple folding and re-folding events occur on the predominant pathway to the native state, with increasing native core interactions and cooperativity as folding progresses.

Theoretical Insight into the Photodegradation of a Disulfide Bridged Cyclic Tetrapeptide in Solution and Subsequent Fast Unfolding−Refolding Events

We report the photoinduced peptide bond (C-N) of an amide unit and S-S bond fission mechanisms of the cyclic tetrapeptide [cyclo(Boc-Cys-Pro-Aib-Cys-OMe)] in methanol solvent by using high-level CASSCF/CASPT2/Amber quantum mechanical/molecular mechanical (QM/MM) calculations. The subsequent energy transport and unfolding-refolding events are characterized by using a semiempirical QM/MM molecular dynamics (MD) simulation methodology that is developed in the present work. In the case of high-energy excitation with <193 nm light, the tetrapeptide molecule in the (1)n pi* surface overcomes two barriers with approximately 10.0 kcal/mol, respectively, and uses energy consumption for breaking the hydrogen bond as well as the N-C bond in the amide unit, ultimately leading to the ground state via a conical intersection of CI (S(NP)/S(0)) by structural changes of an increased N-C distance and a O-C-C angle in the amide unit (a two-dimensional model of the reaction coordinates). Following this point, relaxation to a hot molecule with its original structure in the ground state is the predominant decay channel. A large amount of heat (approximately 110.0 kcal/mol) is initially accumulated in the region of the targeted point of the photoexcitation, and more than 60% of the heat is rapidly dissipated into the solvent on the femtosecond time scale. The relatively slower propagation of heat along the peptide backbone reaches a phase of equilibration within 3 ps. A 300 nm photon of light initiates the relaxation along the repulsive S(sigma sigma)((1)sigma sigma*) state and this decays to the CI (S(sigma sigma)/S(0)) in concomitance with the separation of the disulfide bond. Once cysteinyl radicals are generated, the polar solvent of methanol molecules rapidly diffuses around the radicals, forming a solvent cage and reducing the possibility of close contact in a physical sense. The fast unfolding-refolding event is triggered by S-S bond fission and powered by dramatic thermal motion of the methanol solvent that benefits from heat dissipation. The beta-turn opening (unfolding) can be achieved in about 120 ps without the inclusion of the time associated with the photochemical steps and eventually relaxes to a 3(10)-helix structural architecture (refolding) within 200 ps.

HK97 Maturation Studied by Crystallography and H/2H Exchange Reveals the Structural Basis for Exothermic Particle Transitions

HK97 is an exceptionally amenable system for characterizing major conformational changes associated with capsid maturation in double-stranded DNA bacteriophage. HK97 undergoes a capsid expansion of ∼20%, accompanied by major subunit rearrangements during genome packaging. A previous 3.44-Å-resolution crystal structure of the mature capsid Head II and cryo-electron microscopy studies of other intermediate expansion forms of HK97 suggested that, primarily, rigid-body movements facilitated the maturation process. We recently reported a 3.65-Å-resolution structure of the preexpanded particle form Prohead II (P-II) and found that the capsid subunits undergo significant refolding and twisting of the tertiary structure to accommodate expansion. The P-II study focused on major twisting motions in the P-domain and on refolding of the spine helix during the transition. Here we extend the crystallographic comparison between P-II and Head II, characterizing the refolding events occurring in each of the four major domains of the capsid subunit and their effect on quaternary structure stabilization. In addition, hydrogen/deuterium exchange, coupled to mass spectrometry, was used to characterize the structural dynamics of three distinct capsid intermediates: P-II, Expansion Intermediate, and the nearly mature Head I. Differences in the solvent accessibilities of the seven quasi-equivalent capsid subunits, attributed to differences in secondary and quaternary structures, were observed in P-II. Nearly all differences in solvent accessibility among subunits disappear after the first transition to Expansion Intermediate. We show that most of the refolding is coupled to this transformation, an event associated with the transition from asymmetric to symmetric hexamers.

Role of Spike Protein Endodomains in Regulating Coronavirus Entry

Enveloped viruses enter cells by viral glycoprotein-mediated binding to host cells and subsequent fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated membrane-associated tails. Palmitate adducts are generally required for protein-mediated fusions, but their precise roles in the process are unclear. To obtain additional insights into the S-mediated membrane fusion process, we focused on these acylated carboxyl-terminal intravirion tails. Substituting alanines for the cysteines that are subject to palmitoylation had effects on both S incorporation into virions and S-mediated membrane fusions. In specifically dissecting the effects of endodomain mutations on the fusion process, we used antiviral heptad repeat peptides that bind only to folding intermediates in the S-mediated fusion process and found that mutants lacking three palmitoylated cysteines remained in transitional folding states nearly 10 times longer than native S proteins. This slower refolding was also reflected in the paucity of postfusion six-helix bundle configurations among the mutant S proteins. Viruses with fewer palmitoylated S protein cysteines entered cells slowly and had reduced specific infectivities. These findings indicate that lipid adducts anchoring S proteins into virus membranes are necessary for the rapid, productive S protein refolding events that culminate in membrane fusions. These studies reveal a previously unappreciated role for covalently attached lipids on the endodomains of viral proteins eliciting membrane fusion reactions.

Urea Impedes the Hydrophobic Collapse of Partially Unfolded Proteins

Proteins are denatured in aqueous urea solution. The nature of the molecular driving forces has received substantial attention in the past, whereas the question how urea acts at different phases of unfolding is not yet well understood at the atomic level. In particular, it is unclear whether urea actively attacks folded proteins or instead stabilizes unfolded conformations. Here we investigated the effect of urea at different phases of unfolding by molecular dynamics simulations, and the behavior of partially unfolded states in both aqueous urea solution and in pure water was compared. Whereas the partially unfolded protein in water exhibited hydrophobic collapses as primary refolding events, it remained stable or even underwent further unfolding steps in aqueous urea solution. Further, initial unfolding steps of the folded protein were found not to be triggered by urea, but instead, stabilized. The underlying mechanism of this stabilization is a favorable interaction of urea with transiently exposed, less-polar residues and the protein backbone, thereby impeding back-reactions. Taken together, these results suggest that, quite generally, urea-induced protein unfolding proceeds primarily not by active attack. Rather, thermal fluctuations toward the unfolded state are stabilized and the hydrophobic collapse of partially unfolded proteins toward the native state is impeded. As a result, the equilibrium is shifted toward the unfolded state.