Global Conformational Changes Research Articles

Reaction Coordinate and Thermodynamics of Base Flipping in RNA.

Base flipping is a key biophysical event involved in recognition of various ligands by ribonucleic acid (RNA) molecules. However, the mechanism of base flipping in RNA remains poorly understood, in part due to the lack of atomistic details on complex rearrangements in neighboring bases. In this work, we applied transition path sampling (TPS) methods to study base flipping in a double-stranded RNA (dsRNA) molecule that is known to interact with RNA-editing enzymes through this mechanism. We obtained an ensemble of 1000 transition trajectories to describe the base-flipping process. We used the likelihood maximization method to determine the refined reaction coordinate (RC) consisting of two collective variables (CVs), a distance and a dihedral angle between nucleotides that form stacking interactions with the flipping base. The free energy profile projected along the refined RC revealed three minima, two corresponding to the initial and final states and one for a metastable state. We suggest that the metastable state likely represents a wobbled conformation of nucleobases observed in NMR studies that is often characterized as the flipped state. The analyses of reactive trajectories further revealed that the base flipping is coupled to a global conformational change in a stem-loop of dsRNA.

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

Structural Switches Regulating the Alternating Access Mechanism of Human P-Glycoprotein

P-glycoprotein (Pgp) is a biomedically important ATP-binding cassette (ABC) transporter involved in the development of multidrug resistance in cancer cells. In spite of the canonized work on the transporter function, a lack of general consensus remains concerning the structural details of the different conformational states of Pgp. A major limitation in the study of conformational transitions in ABC transporters is that the process is driven by formation of specific ATP interactions with the nucleotide binding domains (NBDs) of the transporter. Using a novel steered molecular dynamics methodology coupling specific ligand-protein and inter-domain salt-bridge interactions with global conformational changes, we successfully generate four different states of human Pgp, well-separated on the conformational landscape. These include the first stable and catalytically active ATP-bound outward-facing (OF) conformation displaying an open extracellular gate stabilized by lipid binding inside the lumen, as well as an APO (nucleotide-free) inward-facing (IF) conformation showing an open cytoplasmic gate and a large central drug-binding cavity capable of recruiting substrates. In addition, we characterize the only available ATP-bound human Pgp structure as the substrate-free occluded (OC) state of the transporter, with the lumen present in a collapsed state, and likely responsible for the basal-ATPase activity of Pgp. Binding of ATP to the APO IF conformation promotes the closure of the cytoplasmic gate as well as the collapse of the central cavity, likely inducing the transition to the substrate-free OC state. Differential formation of specific salt bridges located at the drug-entry portals in the transmembrane domain as well as between the two NBDs may play an important role in allowing transition between the ATP-bound IF and OF states. The study provides key molecular insights into the specific local switches regulating the global conformational transitions in the transport cycle of human Pgp.

Structural Perspective into Activation of Arp2-3 Complex for Nucleating Linear and Branched Actin Assemblies

Forces generated by actin-filament polymerization are vital for multiple cellular processes. Nucleation of actin-monomers is the very first step in filament assembly and is not a spontaneous process. Nucleator like Arp2/3 complex undergoes conformational changes upon activation by nucleation promoting factors (NPFs) and forms filamentous actin-like seeds to initiate filament assembly. This process is hijacked by pathogens during infection and their deregulation is the underlying cause of various diseases. Though originally discovered as a branched-actin nucleator, more recently Arp2/3 complex was also found to nucleate linear actin-filaments in cells. This variation in nucleating property of Arp2/3 complex has been attributed to activation by different families of NPFs. Contrary to linear actin-filament polymerization by activated Arp2/3 complex, pre-existing actin filament is also required for stimulating Arp2/3 complex to polymerize daughter-filament in branched-actin assemblies. This poses the questions as to how different NPFs alter the nucleating property of Arp2/3 complex to form two different types of actin assemblies, and the role of pre-formed filament in stimulating Arp2/3 complex to polymerize daughter-filament? These have remained unanswered, mainly due to a lack of high-resolution structures of such activated Arp2/3 complex-mediated filament assemblies. Using cryo-electron microscopy, we determined a ∼3.9Å resolution structure of such an assembly. A comparison of this structure with other existing high-resolution structures of Arp2/3 complex reveals differences in local and global conformational changes in the molecule that gives rise to its unique linear and branched-actin filament nucleating properties. Besides delineating the precise interactions between mother-filament and activated Arp2/3 complex in branched-actin networks, we also compare these interactions to those between NPFs and Arp2/3 with the linear-actin filament. This reveals how different activating factors stimulate Arp2/3 complex to initiate both linear and branched actin assemblies.

Structural Investigation into Gating and Modulation of Alpha1 Glycine Receptor in Lipid Nanodiscs

Glycine receptors (GlyR) mediate fast chemical transmission of synaptic signals in the central and peripheral nervous system. GlyR are anionic members of pentameric ligand gated ion channels and are central players in motor coordination and pain perception. Potentiation of GlyR activity results in dampening pain signals in the spinal cord making these receptors an attractive target for pain therapy. Here, we present Cryo-EM structures of the full-length alpha1 GlyR reconstituted into lipid nanodiscs that are captured in multiple functional states that include, the unliganded state (closed) and glycine-bound conformations (open and desensitized). in addition, we present conformations of GlyR in various ligand-bound forms that provide insights into the mechanisms underlying allosteric modulation. The functional state assignments were validated by molecular dynamics simulations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation.

A computational method to simulate global conformational changes of proteins induced by cosolvent.

A computational method to investigate the global conformational change of a protein is proposed by combining the linear response path following (LRPF) method and three-dimensional reference interaction site model (3D-RISM) theory, which is referred to as the LRPF/3D-RISM method. The proposed method makes it possible to efficiently simulate protein conformational changes caused by either solutions of varying concentrations or the presence of cosolvent species by taking advantage of the LRPF and 3D-RISM. The proposed method is applied to the urea-induced denaturation of ubiquitin. The LRPF/3D-RISM trajectories successfully simulate the early stage of the denaturation process within the simulation time of 300 ns, whereas no significant structural change is observed even in the 1 μs standard MD simulation. The obtained LRPF/3D-RISM trajectories reproduce the mechanism of the urea denaturation of ubiquitin reported in previous studies, and demonstrate the high efficiency of the method.

Recombinant expression and purification of pentameric ligand-gated ion channels for Cryo-EM structural studies.

Pentameric ligand-gated ion channels (pLGICs) are central players in synaptic neurotransmission and are targets to a range of drugs used to treat neurological disorders and pain. pLGICs are intrinsically dynamic membrane proteins that upon stimulation by neurotransmitters, undergo global conformational changes across multiple domains spanning a distance of over 165Å. The inter-domain flexibility, a feature crucial for their function as signal transducers in chemical synapses, has been problematic in the efforts toward determining high-resolution structures. Earlier structural studies tackled this issue with a variety of strategies that included partial truncation of flexible domains and the use of antibodies and small-molecule inhibitors to restrict domain movement. With the recent advances in cryo-electron microscopy and single-particle analysis, many of these limitations have been overcome. Here, we describe the methods used in the recombinant expression and purification of full-length constructs of two members of the pentameric ligand-gated ion channel family and the approaches used for capturing multiple conformations in cryo-EM imaging.

Allosteric and transport modulation of human concentrative nucleoside transporter 3 at the atomic scale.

Nucleosides are important precursors of nucleotide synthesis in cells, and nucleoside transporters play an important role in many physiological processes by mediating transmembrane transport and absorption. During nucleoside transport, such proteins undergo a significant conformational transition between the outward- and inward-facing states, which leads to alternating access of the substrate-binding site to either side of the membrane. In this work, a variety of molecular simulation methods have been applied to comparatively investigate the motion modes of human concentrative nucleoside transporter 3 (hCNT3) in three states, as well as global and local cavity conformational changes; and finally, a possible elevator-like transport mechanism consistent with experimental data was proposed. The results of the Gaussian network model (GNM) and anisotropic network model (ANM) show that hCNT3 as a whole tends to contract inwards and shift towards a membrane inside, exhibiting an allosteric process that is more energetically favorable than the rigid conversion. To reveal the complete allosteric process of hCNT3 in detail, a series of intermediate conformations were obtained by an adaptive anisotropic network model (aANM). One of the simulated intermediate states is similar to that of a crystal structure, which indicates that the allosteric process is reliable; the state with lower energy is slightly inclined to the inward-facing structure rather than the expected intermediate crystal structure. The final HOLE analysis showed that except for the outward-facing state, the transport channels were gradually enlarged, which was conductive to the directional transport of nucleosides. Our work provides a theoretical basis for the multistep elevator-like transportation mechanism of nucleosides, which helps to further understand the dynamic recognition between nucleoside substrates and hCNT3 as well as the design of nucleoside anticancer drugs.

CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

Atypical structure and function of integrin αV β8.

Integrins are heterodimeric transmembrane proteins that play important roles in various biological processes. Most integrins serve as adhesion molecules and transmit bidirectional signaling across the cell membrane through global conformational changes from the bent closed to the extended open conformation. However, integrin β8 is distinctive in structure and function. Its cytoplasmic domain lacks the conserved protein-binding sequence, which is important in transmitting inside-out signals, suggesting that integrin β8 may have a different activation mechanism or lack such signaling. In addition, the ligand-binding or activating metal ion Mn2+ does not induce a global conformational change in integrin β8 . It may have only one conformation, that is, an extended, closed conformation, but with high affinity for ligands under physiological conditions, and is, therefore, considered an atypical integrin member. The extended structure and high ligand-binding affinity of integrin αv β8 make it ideal for encountering and binding ligands expressed on an opposing cell or in the extracellular matrix. In this review, we summarize the progress in integrin β8 research with a focus on its distinctive function and structure among integrin members.

Surveying the Side-Chain Network Approach to Protein Structure and Dynamics: The SARS-CoV-2 Spike Protein as an Illustrative Case.

Network theory-based approaches provide valuable insights into the variations in global structural connectivity between different dynamical states of proteins. Our objective is to review network-based analyses to elucidate such variations, especially in the context of subtle conformational changes. We present technical details of the construction and analyses of protein structure networks, encompassing both the non-covalent connectivity and dynamics. We examine the selection of optimal criteria for connectivity based on the physical concept of percolation. We highlight the advantages of using side-chain-based network metrics in contrast to backbone measurements. As an illustrative example, we apply the described network approach to investigate the global conformational changes between the closed and partially open states of the SARS-CoV-2 spike protein. These conformational changes in the spike protein is crucial for coronavirus entry and fusion into human cells. Our analysis reveals global structural reorientations between the two states of the spike protein despite small changes between the two states at the backbone level. We also observe some differences at strategic locations in the structures, correlating with their functions, asserting the advantages of the side-chain network analysis. Finally, we present a view of allostery as a subtle synergistic-global change between the ligand and the receptor, the incorporation of which would enhance drug design strategies.

Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.

The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and by the opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane-interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the "late" 135S particles have detectable levels of the VP1 N-terminus been trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously.

A Computational Analysis of the Factors Governing the Dynamics of α7 nAChR and Its Homologs

The α7 nicotinic acetylcholine receptor is a homopentameric ion channel from the Cys-loop receptor superfamily targeted for psychiatric indications and inflammatory pain. Molecular dynamics studies of the receptor have focused on residue mobility and global conformational changes to address receptor function. However, a comparative analysis of α7 with its homologs that cannot trigger channel opening has not been made so far. To identify the residues involved in α7 activation, we ran triplicate 500-ns molecular dynamics simulations with an α7 extracellular domain homology model and two acetylcholine-binding protein homologs. We tested the effect of ligand binding and amino acid sequence on the structure and dynamics of the three proteins. We found that mobile regions identified based on root mean-square deviation and root mean-square fluctuation values are not always consistent among the individual α7 extracellular domain simulations. Comparison of the replica-average properties of the three proteins based on dynamic cross-correlation maps showed that ligand binding affects the coupling between the C-loop and the Cys-loop, vestibular loop, and β1–β2 loops. In addition, the main-immunogenic-region-like domain of α7 went through correlated motions with multiple domains of the receptor. These correlated motions were absent or diminished in α7 homologs, suggesting a unique role in α7 activation.

Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state.

Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.

Key role of quinone in the mechanism of respiratory complex I

Complex I is the first and the largest enzyme of respiratory chains in bacteria and mitochondria. The mechanism which couples spatially separated transfer of electrons to proton translocation in complex I is not known. Here we report five crystal structures of T. thermophilus enzyme in complex with NADH or quinone-like compounds. We also determined cryo-EM structures of major and minor native states of the complex, differing in the position of the peripheral arm. Crystal structures show that binding of quinone-like compounds (but not of NADH) leads to a related global conformational change, accompanied by local re-arrangements propagating from the quinone site to the nearest proton channel. Normal mode and molecular dynamics analyses indicate that these are likely to represent the first steps in the proton translocation mechanism. Our results suggest that quinone binding and chemistry play a key role in the coupling mechanism of complex I.

Abstract 293: Heat shock factor 1 directly suppresses AMP-activated protein kinase to promote tumorigenesis

Abstract Heat shock factor 1 (HSF1), a master transcriptional regulator of the evolutionarily conserved proteotoxic stress response, preserves proteomic stability upon environmental insults. HSF1 is also a potent pro-oncogenic factor, suggesting that proteomic stability enables oncogenesis. AMP-activated protein kinase (AMPK), a key cellular metabolic sensor and downstream effector of the tumor suppressor LKB1, activates catabolic pathways but suppresses anabolic pathways, including fatty acid and cholesterol biosynthesis, thereby maintaining energy homeostasis. We previously showed that upon activation by metabolic stressors, AMPK interacts with and phosphorylates HSF1 to inactivate it and cause proteomic instability, leading to tumor suppression. Intriguingly, we also discovered that HSF1 suppresses AMPK and controls body fat mass. This study is to investigate the mechanisms by which HSF1 reciprocally suppresses AMPK and the roles of this suppression in lipid metabolism and tumorigenesis. By utilizing recombinant proteins, HSF1 peptide libraries and transcription-deficient HSF1 mutants, our in vitro studies reveal that through physical interactions HSF1 imposes multilayer regulations on AMPK, including blocking AMP binding to γ subunits, impairing LKB1-mediated Thr172 phosphorylation, promoting Thr172 de-phosphorylation by PP2A, and impeding ATP binding to catalytic α subunits. Results from circular dichroism spectroscopy reveal that HSF1 induces global AMPK conformational changes. Biologically, Hsf1 deficiency suppresses lipogenesis and decreases lipid content via AMPK activation. Moreover, Hsf1-deficient cells and mice display reduced cholesterol levels. Interestingly, this defect leads to impaired cholesteroylation of sonic hedgehog (SHH). Consequently, the oncogenic SHH signaling is impaired in HSF1-deficient cells. In vivo, Hsf1 deficiency reduces body fat mass in mice, which can be markedly rescued by either pharmacological or genetic inhibition of AMPK. Importantly, the transcription-deficient HSF1 mutant, through AMPK suppression, enhances the lipid content and SHH cholesteroylation, and promotes the in vivo growth of xenografted human melanomas. In conclusion, HSF1 is a direct AMPK antagonist. This transcription-independent interaction of HSF1 with AMPK epitomizes a reciprocal kinase-substrate regulation whereby lipid metabolism and proteomic stability intertwine and promotes cancer anabolism and oncogenesis. Citation Format: Kuo-Hui Su, Siyuan Dai, Zijian Tang, Meng Xu, Chengkai Dai. Heat shock factor 1 directly suppresses AMP-activated protein kinase to promote tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 293.

Mechanisms of activation and desensitization of full-length glycine receptor in lipid nanodiscs

Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.

Interaction of N-succinyl diaminopimelate desuccinylase with orphenadrine and disulfiram

The emergence of multiresistant, often persistent, pathogenic bacteria emphasizes the need for a continuous identification of new pharmacological targets of enzymatic nature, and the development of selective inhibitors against them. Ubiquitously present in most bacteria, the enzyme metallohydrolase N-succinyl diaminopimelate desuccinylase (DapE) is required for the biosynthesis of meso-diaminopimelate (mDAP) and lysine, both essential components of bacterial peptidoglycan. DapE activity has been recognized as critical for bacterial growth; and thus, it is a potential pharmacological target. In order to develop effective inhibitors against DapE, understanding of structural and functional features of the enzyme must be used in the design, such as the interaction of its metal centers with ligands, as well as its effect on global protein conformational changes that seem to produce an induced fit after ligand binding. Here, we propose the potential application of currently approved drugs, used in different medical fields, orphenadrine and disulfiram, as possible inhibitory compounds against DapE, based on studies of equilibrium ligand binding, inhibition, thermal stability and molecular docking into Enterococcus faecium and Escherichia coli DapE enzyme homologs. Drugs were selected based on key structural features, including the presence of soft heteroatoms or π-bonds that are known to interact with DapE active site. Enzymes from selected bacteria were chosen based on the pattern of infection, persistence, and drug resistance as well to study an enzyme from the two Gram classification. Furthermore, the information presented here can further provide structural details about key interacting functional groups, which should be considered in the design and development of a new generation of antibiotics that can target the essential DapE enzyme.

Impact of Silane Monolayers on the Adsorption of Streptavidin on Silica and Its Subsequent Interactions with Biotin: Molecular Dynamics and Steered Molecular Dynamics Simulations.

Protein adsorption on surfaces is used in analytical tools as an immobilization mean to trap the analyte to be detected. However, protein adsorption can lead to a conformational change in the protein structure, resulting in a loss of bioactivity. Here, we study adsorption of a streptavidin-biotin complex on amorphous SiO2 surfaces functionalized with five different silane self-assembled monolayers by all-atom molecular dynamics simulations. We find that the streptavidin global conformational change, as well as the nature of residues with high mobility, depends on the alkyl chain length and head-group charge of silane molecules. Effects on interactions with biotin are further investigated by steered molecular dynamics (SMD) simulations, which mimics atomic force microscopy (AFM) with the biotin attached on the tip. We show the combined effects of adsorption-induced global conformational changes and of the position of residues with high mobility on the streptavidin-biotin rupture force. By comparing our results to experimental and SMD rupture forces obtained in water, without any surface, we conclude that silane with uncharged and short alkyl chains allows streptavidin immobilization, while keeping biotin interactions better than silanes with long alkyl chains or charged head groups.

Protein folding from heterogeneous unfolded state revealed by time-resolved X-ray solution scattering

One of the most challenging tasks in biological science is to understand how a protein folds. In theoretical studies, the hypothesis adopting a funnel-like free-energy landscape has been recognized as a prominent scheme for explaining protein folding in views of both internal energy and conformational heterogeneity of a protein. Despite numerous experimental efforts, however, comprehensively studying protein folding with respect to its global conformational changes in conjunction with the heterogeneity has been elusive. Here we investigate the redox-coupled folding dynamics of equine heart cytochrome c (cyt-c) induced by external electron injection by using time-resolved X-ray solution scattering. A systematic kinetic analysis unveils a kinetic model for its folding with a stretched exponential behavior during the transition toward the folded state. With the aid of the ensemble optimization method combined with molecular dynamics simulations, we found that during the folding the heterogeneously populated ensemble of the unfolded state is converted to a narrowly populated ensemble of folded conformations. These observations obtained from the kinetic and the structural analyses of X-ray scattering data reveal that the folding dynamics of cyt-c accompanies many parallel pathways associated with the heterogeneously populated ensemble of unfolded conformations, resulting in the stretched exponential kinetics at room temperature. This finding provides direct evidence with a view to microscopic protein conformations that the cyt-c folding initiates from a highly heterogeneous unfolded state, passes through still diverse intermediate structures, and reaches structural homogeneity by arriving at the folded state.