Folding Kinetics Research Articles

A PDZ tandem repeat folds and unfolds via different pathways

AbstractProtein folding and unfolding experiments are interpreted under the assumption of microscopic reversibility, that is, that at equilibrium one process is the reverse of the other. Single‐domain proteins illustrate the validity of such an interpretation, although reversibility does not necessarily hold under the different conditions typically used for folding and unfolding experiments. In fact, more complex proteins, which often exhibit irreversible unfolding, are generally considered not amenable to folding kinetics studies. Here, the X11 PDZ1‐PDZ2 tandem repeat allows us to reveal the different folding and unfolding pathways at play under different experimental conditions, thus reconciling the apparent contradiction between theory and experiment.

Sustaining 500,000 Folding Cycles Through Bioinspired Stress Dispersion Design in Sodium‐Ion Batteries

AbstractDeveloping super‐foldable electronic materials and devices presents a significant challenge, as intrinsic conductive materials are unable to achieve numerous true‐folding operations (super‐foldable) due to limitations from short‐range forces of chemical bonds. Consequently, super‐foldable batteries remain unexplored. This work focused on sodium‐ion batteries as a breakthrough point to advance super‐foldable devices. By employing a “2+1” bioinspired strategy, we stepwise designed and assembled super‐foldable components, from substrates to electrodes, and to ultimately device. This bioinspired approach completely disperses folding stress and thus prevents the breakage of chemical bonds, enabling the successful fabrication of the first super‐foldable ion battery. This battery can withstand true‐folding at any angle, in any direction, and for an unprecedented number of cycles—far outperforming current foldable phones with hinge structures. Remarkably, after 500,000 true‐folding cycles, the battery's microstructure remains intact with no significant degradation of electrochemical performance. Real‐time dynamic folding observations reveal an M‐shaped folding structure within the bioinspired materials, which effectively disperses stress via bulged layers, dispersed arcs, and slidable microgrooves that work together across different directions and dimensions to achieve super‐foldability. Mechanical simulations vividly verify this principle. This work represents a breakthrough in super‐foldable devices, offering valuable insights and promoting practical application for future super‐foldable devices.

Sustaining 500,000 Folding Cycles Through Bioinspired Stress Dispersion Design in Sodium-Ion Batteries.

Developing super-foldable electronic materials and devices presents a significant challenge, as intrinsic conductive materials are unable to achieve numerous true-folding operations (super-foldable) due to limitations from short-range forces of chemical bonds. Consequently, super-foldable batteries remain unexplored. This work focused on sodium-ion batteries as a breakthrough point to advance super-foldable devices. By employing a "2+1" bioinspired strategy, we stepwise designed and assembled super-foldable components, from substrates to electrodes, and to ultimately device. This bioinspired approach completely disperses folding stress and thus prevents the breakage of chemical bonds, enabling the successful fabrication of the first super-foldable ion battery. This battery can withstand true-folding at any angle, in any direction, and for an unprecedented number of cycles-far outperforming current foldable phones with hinge structures. Remarkably, after 500,000 true-folding cycles, the battery's microstructure remains intact with no significant degradation of electrochemical performance. Real-time dynamic folding observations reveal an M-shaped folding structure within the bioinspired materials, which effectively disperses stress via bulged layers, dispersed arcs, and slidable microgrooves that work together across different directions and dimensions to achieve super-foldability. Mechanical simulations vividly verify this principle. This work represents a breakthrough in super-foldable devices, offering valuable insights and promoting practical application for future super-foldable devices.

Improved Estimates of Folding Stabilities and Kinetics with Multiensemble Markov Models.

Markov State Models (MSMs) have been widely applied to understand protein folding mechanisms by predicting long time scale dynamics from ensembles of short molecular simulations. Most MSM estimators enforce detailed balance, assuming that trajectory data are sampled at an equilibrium. This is rarely the case for ab initio folding studies, however, and as a result, MSMs can severely underestimate protein folding stabilities from such data. To remedy this problem, we have developed an enhanced-sampling protocol in which (1) unbiased folding simulations are performed and sparse tICA is used to obtain features that best capture the slowest events in folding, (2) umbrella sampling along this reaction coordinate is performed to observe folding and unfolding transitions, and (3) the thermodynamics and kinetics of folding are estimated using multiensemble Markov models (MEMMs). Using this protocol, folding pathways, rates, and stabilities of a designed α-helical hairpin, Z34C, can be predicted in good agreement with experimental measurements. These results indicate that accurate simulation-based estimates of absolute folding stabilities are within reach, with implications for the computational design of folded miniproteins and peptidomimetics.

Energy landscape of conformational changes for a single unmodified protein

Resolving the free energy landscapes that govern protein biophysics has been obscured by ensemble averaging. While the folding dynamics of single proteins have been observed using fluorescent labels and/or tethers, a simpler and more direct measurement of the conformational changes would not require modifications to the protein. We use nanoaperture optical tweezers to resolve the energy landscape of a single unmodified protein, Bovine Serum Albumin (BSA), and quantify changes in the three-state conformation dynamics with temperature. A Markov model with Kramers’ theory transition rates is used to model the dynamics, showing good agreement with the observed state transitions. This first look at the intrinsic energy landscape of proteins provides a transformative tool for protein biophysics and may be applied broadly, including mapping out the energy landscape of particularly challenging intrinsically disordered proteins.

Kinetics of i-motif folding within the duplex context

Non-canonical nucleic acid structures possess an ability to interact selectively with proteins, thereby exerting influence over various intracellular processes. Numerous studies indicate that genomic G-quadruplexes and i-motifs are involved in the regulation of transcription. These structures are formed temporarily during the unwinding of the DNA double helix; and their direct determination is a rather difficult task. In addition, i-motif folding is pH-dependent, with most i-motifs having low stability at neutral pH. However, some genomic i-motifs with long cytosine repeats were shown to be stable at pH 7.3, suggesting their functionality within the nucleus. Here we studied pH-dependent behavior of a model i-motif with flanking sequences that forms a duplex motif. Kinetic studies on bimodular structures with cytosine residues replaced with an environment-sensitive fluorescent label reveal the stabilization of the i-motif structure near the i-motif-duplex junction. These results highlight the importance of the natural environment of i-motifs for the correct assessment of their stability.

Adaptive CVgen: Leveraging reinforcement learning for advanced sampling in protein folding and chemical reactions

Enhanced sampling techniques have traditionally encountered two significant challenges: identifying suitable reaction coordinates and addressing the exploration-exploitation dilemma, particularly the difficulty of escaping local energy minima. Here, we introduce Adaptive CVgen, a universal adaptive sampling framework designed to tackle these issues. Our approach utilizes a set of collective variables (CVs) to comprehensively cover the system's potential evolutionary phase space, generating diverse reaction coordinates to address the first challenge. Moreover, we integrate reinforcement learning strategies to dynamically adjust the generated reaction coordinates, thereby effectively balancing the exploration-exploitation dilemma. We apply this framework to sample the conformational space of six proteins transitioning from completely disordered states to folded states, as well as to model the chemical synthesis process of C60, achieving conformations that perfectly match the standard C60 structure. The results demonstrate Adaptive CVgen's effectiveness in exploring new conformations and escaping local minima, achieving both sampling efficiency and exploration accuracy. This framework holds potential for extending to various related challenges, including protein folding dynamics, drug targeting, and complex chemical reactions, thereby opening promising avenues for application in these fields.

Optimal strategy for stabilizing protein folding intermediates.

To manipulate the protein concentration at a certain functional state through chemical stabilizers is crucial for protein-related studies. It not only plays a key role in protein structure analysis and protein folding kinetics, but also affects protein functionality to a large extent and thus has wide applications in medicine, food industry, etc. However, due to concerns about side effects or financial costs of stabilizers, identifying optimal strategies for enhancing protein stability with a minimal amount of stabilizers is of great importance. Here, we prove that either for the fixed terminal time (including both finite and infinite cases) or for the free one, the optimal control strategy for stabilizing the folding intermediates with a linear strategy for stabilizer addition belongs to the class of bang-bang controls. The corresponding optimal switching time is derived analytically, whose phase diagram with respect to several key parameters is explored in detail. The bang-bang control will be broken when nonlinear strategies for stabilizer addition are adopted. Moreover, the above theory is applied to the stabilization of erythropoietin by ten different kinds of chemicals, providing theoretical guidance for the selection and rational usage of stabilizers. Our current study on optimal strategies for protein stabilizers not only offers deep insights into the general picture of protein folding kinetics but also provides valuable theoretical guidance on treatments for protein-related diseases in medicine.

‘Invisible’ Molecular Dynamics Revealed for a Conformationally Chiral π‐Stacked Perylene Bisimide Foldamer

Whilst energetic and kinetic aspects of folding processes are meanwhile well understood for natural biomacromolecules, the folding dynamics in so far studied artificial foldamer counterparts remain largely unexplored. This is due to the low energy barriers between their conformational isomers that make the dynamic processes undetectable with conventional methods such as UV/vis absorption, fluorescence, and NMR spectroscopy, making such processes ‘invisible’. Here we present an asymmetric perylene bisimide dimer (bis‐PBI 1) that possesses conformational chirality in its folded state. Owing to the large interconversion barrier (≥ 116 kJ mol–1), four stereoisomers could be separated and isolated. Since the interconversion between these stereoisomers requires the foldamer to first open and then to re‐fold, the transformation of one stereoisomer into others allowed us to ‘visualize’ the dynamics of folding with time and determine its lifetimes and the energetic barriers associated with the folding process. Supported by quantum chemical calculations, we identified the open structure to be only a fleeting metastable state of higher energy. Our experimental observation of the kinetics associated with the molecular dynamics in the PBI foldamer advances the fundamental understanding of folding in synthetic foldamers and paves the way for the design of smart functional materials.

'Invisible' Molecular Dynamics Revealed for a Conformationally Chiral π-Stacked Perylene Bisimide Foldamer.

Whilst energetic and kinetic aspects of folding processes are meanwhile well understood for natural biomacromolecules, the folding dynamics in so far studied artificial foldamer counterparts remain largely unexplored. This is due to the low energy barriers between their conformational isomers that make the dynamic processes undetectable with conventional methods such as UV/vis absorption, fluorescence, and NMR spectroscopy, making such processes 'invisible'. Here we present an asymmetric perylene bisimide dimer (bis-PBI 1) that possesses conformational chirality in its folded state. Owing to the large interconversion barrier (≥ 116 kJ mol-1), four stereoisomers could be separated and isolated. Since the interconversion between these stereoisomers requires the foldamer to first open and then to re-fold, the transformation of one stereoisomer into others allowed us to 'visualize' the dynamics of folding with time and determine its lifetimes and the energetic barriers associated with the folding process. Supported by quantum chemical calculations, we identified the open structure to be only a fleeting metastable state of higher energy. Our experimental observation of the kinetics associated with the molecular dynamics in the PBI foldamer advances the fundamental understanding of folding in synthetic foldamers and paves the way for the design of smart functional materials.

Folding of N-terminally acetylated α-synuclein upon interaction with lipid membranes

α-Synuclein (α-syn) is an abundant presynaptic neuronal protein whose aggregation is strongly associated with Parkinson’s disease. It has been proposed that lipid membranes significantly affect α-syn’s aggregation process. Extensive studies have been conducted to understand the interactions between α-syn and lipid membranes and have demonstrated that the N-terminus plays a critical role. However, the dynamics of the interactions and the conformational transitions of the N-terminus of α-syn at the atomistic scale details are still highly desired. In this study, we performed extensive enhanced sampling molecular dynamics simulations to quantify the folding and interactions of wild-type and N-terminally acetylated α-syn when interacting with lipid structures. We found that N-terminal acetylation significantly increases the helicity of the first few residues in solution or when interacting with lipid membranes. The observations in simulations showed that the binding of α-syn with lipid membranes mainly follows the induced-fit model, where the disordered α-syn binds with the lipid membrane through the electrostatic interactions and hydrophobic contacts with the packing defects; after stable insertion, N-terminal acetylation promotes the helical folding of the N-terminus to enhance the anchoring, thus increasing the binding affinity. We have shown the critical role of the first N-terminal residue methionine for recognition and anchoring to the negatively charged membrane. Although N-terminal acetylation neutralizes the positive charge of Met1 that may affect the electrostatic interactions of α-syn with membranes, the increase in helicity of the N-terminus should compensate for the binding affinity. This study provides detailed insight into the folding dynamics of α-syn’s N-terminus with or without acetylation in solution and upon interaction with lipids, which clarifies how the N-terminal acetylation regulates the affinity of α-syn binding to lipid membranes. It also shows how packing defects and electrostatic effects coregulate the N-terminus of α-syn folding and its interaction with membranes.

Modulation of dynamic DNA G-quadruplex structures in the hTERT promoter region by ligands.

Small molecules can inhibit cellular processes such as replication and transcription by binding to the promoter regions that are prone to form G-quadruplexes. However, since G-quadruplexes exist throughout the human genome, the G-quadruplex binders suffer from specificity issues. To tackle this problem, a G-quadruplex binder (Pyridostatin, or PDS) is conjugated with a ligand (Polyamide, or PA) that can specifically recognize DNA sequences flanking the G-quadruplex forming region. The binding mechanism of this hybrid ligand to the hTERT promoter region (hTERT 5-12) is then elucidated using optical tweezers. During mechanical unfolding processes, different intermediate structures of hTERT 5-12 in presence of PDS, PA, or PA-PDS conjugateare observed. These intermediate structures are consistent with two folding patterns of G-quadruplexes in the hTERT 5-12 fragment. While the duplex DNA binder PA facilitates the folding of a hairpin-G-quadruplex structure, the PDS assists the formation of two tandem G-quadruplexes. Both replication stop assay in vitro and dual luciferase assay in vivo established the effectiveness of the PA-PDS conjugate for hTERT 5-12 targeting. We expect such a ligand dependent folding dynamics will provide guidelines to the development of drugs that not only target hTERT expressions, but also other oncogenes via interactions with specific G-quadruplex structures formed in their promotor regions.

N-terminal domain of polypyrimidine-tract binding protein is a dynamic folding platform for adaptive RNA recognition.

The N-terminal RNA recognition motif domain (RRM1) of polypyrimidine tract binding protein (PTB) forms an additional C-terminal helix α3, which docks to one edge of the β-sheet upon binding to a stem-loop RNA containing a UCUUU pentaloop. Importantly, α3 does not contact the RNA. The α3 helix therefore represents an allosteric means to regulate the conformation of adjacent domains in PTB upon binding structured RNAs. Here we investigate the process of dynamic adaptation by stem-loop RNA and RRM1 using NMR and MD in order to obtain mechanistic insights on how this allostery is achieved. Relaxation data and NMR structure determination of the free protein show that α3 is partially ordered and interacts with the domain transiently. Stem-loop RNA binding quenches fast time scale dynamics and α3 becomes ordered, however microsecond dynamics at the protein-RNA interface is observed. MD shows how RRM1 binding to the stem-loop RNA is coupled to the stabilization of the C-terminal helix and helps to transduce differences in RNA loop sequence into changes in α3 length and order. IRES assays of full length PTB and a mutant with altered dynamics in the α3 region show that this dynamic allostery influences PTB function in cultured HEK293T cells.

The importance of the location of the N-terminus in successful protein folding in vivo and in vitro

Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors.

Self‐Adaptive Synthesis of Non‐Covalent Crosslinkers while Folding Single‐Chain Polymers

AbstractPeptide folding is a dynamic process driven by non‐covalent cross‐linking leading to functional nanostructures for essential biochemical activities. However, replicating this process in synthetic systems is challenging due to the difficulty in mimicking nature‘s real‐time regulation of non‐covalent crosslinking for single‐chain polymer folding. Here, we address this by employing anionic dithiol building blocks to create macrocyclic disulfides as non‐covalent crosslinkers that adapted to the folding process. Initially, small macrocycles facilitated a low degree folding of a polycation. Then, this preorganized structure catalysed the production of larger macrocycles that enhanced the folding conversely. The self‐adaptive synthesis was verified through the encapsulation of an anticancer drug, showing an updated production distribution of non‐covalent crosslinkers and maximizing drug‐loading efficiency against drug‐resistant cancer in vitro. Our research advances the understanding of molecular systems by exploring species evolution via the structural dynamics of polymer folding. Additionally, adaptive synthesis enables controlled, sequential folding of synthetic polymers, with the potential to mimic protein functions.

Self-Adaptive Synthesis of Non-Covalent Crosslinkers while Folding Single-Chain Polymers.

Peptide folding is a dynamic process driven by non-covalent cross-linking leading to functional nanostructures for essential biochemical activities. However, replicating this process in synthetic systems is challenging due to the difficulty in mimicking nature's real-time regulation of non-covalent crosslinking for single-chain polymer folding. Here, we address this by employing anionic dithiol building blocks to create macrocyclic disulfides as non-covalent crosslinkers that adapted to the folding process. Initially, small macrocycles facilitated a low degree folding of a polycation. Then, this preorganized structure catalysed the production of larger macrocycles that enhanced the folding conversely. The self-adaptive synthesis was verified through the encapsulation of an anticancer drug, showing an updated production distribution of non-covalent crosslinkers and maximizing drug-loading efficiency against drug-resistant cancer in vitro. Our research advances the understanding of molecular systems by exploring species evolution via the structural dynamics of polymer folding. Additionally, adaptive synthesis enables controlled, sequential folding of synthetic polymers, with the potential to mimic protein functions.

Chemical Synthesis and Structure-Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata.

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure-activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen-Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles.

Upper blepharoplasty in Asian population: A novel technique based on functional zoning and dynamic reconstruction with anatomical structure.

Double eyelid blepharoplasty is the most popular cosmetic operation in Asian population. Various surgical techniques have been developed in order to create dynamic eyelid folds with natural-looking in recent years, but postoperative complications like so-called sausage-like appearance have not been resolved completely. To create natural-looking dynamic folds, we propose a new method imitating the original anatomical structure of congenital double-eyelid. Eighty-six patients who underwent the double-eyelid surgery from June 1st, 2018 to October 31st, 2020 were included in this retrospective study, including 10 males and 76 females, aged 18-39 years (mean 27.4 ± 5.6 years). All the included patients received double eyelid surgery performed by the same senior doctor, using the pretarsal orbicularis oculi muscle-releasing technique. Patient Reported Outcome Measures questionnaires were administrated to assess the severity of scarring, pain, and asymmetry, as well as functional and appearance issues. Surgical outcome was assessed through objective and subjective evaluation forms (PROM and patient satisfaction rate). Among the 86 patients, 5 were lost during the follow-up period. The absolute number of enrolled patients is 81. 91.36% of the enrolled patients reported minimal or non-visible scarring at the double eyelid incision. As to functional and appearance issues, the main problem were asymmetry (12.35%) and the narrowing of the supratarsal crease width (8.64%). No supratarsal depression and "sausage-like" appearance occurred in this study. 95.1% of patients reported either good or excellent outcome (mean score: 108 of 120) based on analysis of PROM results, and 96.3% of patients reported either high or very high satisfaction (mean score: 96 of 120) for the patient satisfaction assessment. This new surgical method of double-eyelid blepharoplasty provides comparatively safe and effective results.

FACT mediates the depletion of macroH2A1.2 to expedite gene transcription

The histone variant macroH2A is generally linked to transcriptionally inactive chromatin, but how macroH2A regulates chromatin structure and functions in the transcriptional process remains elusive. This study reveals that while the integration of human macroH2A1.2 into nucleosomes does not affect their stability or folding dynamics, it notably hinders the maintenance of facilitates chromatin transcription's (FACT's) function. We show that FACT effectively diminishes the stability of macroH2A1.2-nucleosomes and expedites their depletion subsequent to the initial unfolding process. Furthermore, we identify the residue S139 in macroH2A1.2 as a critical switch to modulate FACT's function in nucleosome maintenance. Genome-wide analyses demonstrate that FACT-mediated depletion of macroH2A-nucleosomes allows the correct localization of macroH2A, while the S139 mutation reshapes macroH2A distribution and influences stimulation-induced transcription and cellular response in macrophages. Our findings provide mechanistic insights into the intricate interplay between macroH2A and FACT at the nucleosome level and elucidate their collective role in transcriptional regulation and immune response of macrophages.

Electrochemical aptasensor based on RGO/PPy/Au*NPs for diagnostic biosensing of SARS-CoV-2 in clinical samples: Experimental and molecular dynamics simulations approaches

A highly sensitive and selective E-DNA biosensor was fabricated on a gold substrate using a reduced graphene oxide/polypyrrole/gold nanoparticle/oligonucleotide (RGO/PPy/Au*NPs/Apt) nanocomposite electrode to detect the nucleocapsid protein of SARS-CoV-2 in the patient blood plasma. The modified electrode was characterized by physicochemical techniques such as Fourier transform-infra red (FT-IR), Raman spectroscopy, X-ray diffraction (XRD), energy-dispersive X-ray (EDX) spectroscopy, X-ray photoelectron spectroscopy (XPS) analyses, Brunauer-Emmett-Teller (BET), and Barrett-Joyner-Halenda (BJH) analyses. Moreover, electrochemical analyses were employed to study the electrochemical performance of the electrodes including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV). Computational investigations of N protein bindings such as hydrogen bonding, Van der Waals binding, and Gibbs binding energies of the N-protein, and aptamer were studied by Molecular Dynamics Simulations (MDS). The unique synergistic effect of RGO, PPy, and the well-known effect of Au nanoparticles makes the DNA probe immobilized on the gold electrode surface. After optimizing the systems, the E-DNA biosensor exhibited a fast SWV response, higher sensitivity (33.77 μA.nM−1.cm−2) and selectivity, high efficiency, good storage stability, and acceptable repeatability for monitoring DNA. The results of real samples based on SWV indicated the correct functioning of the aptasensor in the presence of the SARS-CoV-2 virus. The limit of detection was 3.16×10−17 M and the limit of quantitation was 1.42×10−16 M. The MDS results indicated the stable dynamic folding of the aptamer for beneficial binding. The results indicated that the RGO/PPy/Au*NPs/Apt biosensor is promising for detecting of SARS-CoV-2 virus.