Folding Kinetics Research Articles

Effect of U-Flex-to-Install and Dynamic U-Flexing on Li-Ion Battery State of Health Degradation Subjected to Varying Fold Orientations, Folding Speeds, Depths of Charge, C-Rates, and Temperatures

Abstract The demand for wearable consumer electronics, fitness accessories, and biomedical equipment has led to the growth research and development of thin flexible batteries. Wearable equipment and other asset monitoring applications require conformal installation of power sources on nonplanar surfaces. For power sources in wearable electronics, durability to sustain repetitive mechanical stresses induced by human body motion is paramount along with the usual desirable power source characteristics. Previous research documenting the reliability of statically and dynamically folded power sources is scarce and does not follow standardized test protocols. Particularly, the use of manual stressing for mechanical folding of the power sources instead of a mechanical test setup is a key shortcoming in existing literature. Data are lacking on battery life cycling and in situ mechanical stress-testing of the power sources including their impact of performance and reliability. This study aims to overcome these deficiencies by testing a commercial Li-ion power source under static as well as dynamic folding. Furthermore, the fold orientation and its fold speed are varied to evaluate the effect of different mechanical stress topologies on the power source. Finally, a regression model was developed to capture the effect of these use parameters on battery capacity degradation.

Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1

Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The ΔF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the ΔF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the ΔF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. We found that the NBD1 α-subdomain with three adjacent strands from the β-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this α-core region and increases the resistance of the ΔF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors.

Transactivation domain of Adenovirus Early Region 1A (E1A): Investigating folding dynamics and aggregation.

Transactivation domain of Adenovirus Early region 1A (E1A) oncoprotein is an intrinsically disordered molecular hub protein. It is involved in binding to different domains of human cell transcriptional co-activators such as retinoblastoma (pRb), CREB-binding protein (CBP), and its paralogue p300. The conserved region 1 (TAD) of E1A is known to undergo structural transitions and folds upon interaction with transcriptional adaptor zinc finger 2 (TAZ2). Previous reports on Taz2-E1A studies have suggested the formation of helical conformations of E1A-TAD. However, the folding behavior of the TAD region in isolation has not been studied in detail. Here, we have elucidated the folding behavior of E1A peptide at varied temperatures and solution conditions. Further, we have studied the effects of macromolecular crowding on E1A-TAD peptide. Additionally, we have also predicted the molecular recognition features of E1A using MoRF predictors. The predicted MoRFs are consistent with its structural transitions observed during TAZ2 interactions for transcriptional regulation in literature. Also, as a general rule of MoRFs, E1A undergoes helical transitions in alcohol and osmolyte solution. Finally, we studied the aggregation behavior of E1A, where we observed that the E1A could form amyloid-like aggregates that are cytotoxic to mammalian cells.

Instantaneous splicing and excision of inteins to synthesize polyproteins on a substrate with tunable linkers.

Nature has adapted chimeric polyproteins to achieve superior and multiplexed functionality in a single protein. However, the hurdles in in vitro synthesis have restricted the biomimicry of and subsequent fundamental studies on the structure-function relationship of polyproteins. Recombinant expression of polyproteins and the synthesis of polyproteins via the enzyme-mediated repetitive digestion and ligation of individual protein domains have been widely practiced. However, recombinant expression often suffers from an in vitro refolding process, whereas enzyme-assisted peptide conjugation results in heterogeneous products, primarily due to enzymatic re-digestion, and prolonged and multistep reactions. Moreover, both methods incorporate enzyme-recognition residues of varying lengths as artifacts at interdomain linkers. The linkers, although tiny, regulate the spatiotemporal conformations of the polyproteins differentially and tune the folding dynamics, stability, and functions of the constituent protein. In an attempt to leave no string behind at the interdomain junctions, here, we develop a 'splice and excise' synthetic route for polyproteins on a substrate using two orthogonal split inteins. Inteins self-excise and conjugate the protein units covalently and instantaneously, without any cofactors, and incorporate a single cysteine or serine residue at the interdomain junctions.

Shape variability of the central sulcus in the developing brain: A longitudinal descriptive and predictive study in preterm infants

Despite growing evidence of links between sulcation and function in the adult brain, the folding dynamics, occurring mostly before normal-term-birth, is vastly unknown. Looking into the development of cortical sulci in infants can give us keys to address fundamental questions: what is the sulcal shape variability in the developing brain? When are the shape features encoded? How are these morphological parameters related to further functional development?In this study, we aimed to investigate the shape variability of the developing central sulcus, which is the frontier between the primary somatosensory and motor cortices. We studied a cohort of 71 extremely preterm infants scanned twice using MRI – once around 30 weeks post-menstrual age (w PMA) and once at term-equivalent age, around 40w PMA –, in order to quantify the sulcus's shape variability using manifold learning, regardless of age-group or hemisphere. We then used these shape descriptors to evaluate the sulcus's variability at both ages and to assess hemispheric and age-group specificities. This led us to propose a description of ten shape features capturing the variability in the central sulcus of preterm infants. Our results suggested that most of these features (8/10) are encoded as early as 30w PMA. We unprecedentedly observed hemispheric asymmetries at both ages, and the one captured at term-equivalent age seems to correspond with the asymmetry pattern previously reported in adults. We further trained classifiers in order to explore the predictive value of these shape features on manual performance at 5 years of age (handedness and fine motor outcome). The central sulcus's shape alone showed a limited but relevant predictive capacity in both cases. The study of sulcal shape features during early neurodevelopment may participate to a better comprehension of the complex links between morphological and functional organization of the developing brain.

Observing the base-by-base search for native structure along transition paths during the folding of single nucleic acid hairpins

Biomolecular folding involves searching among myriad possibilities for the native conformation, but the elementary steps expected from theory for this search have never been detected directly. We probed the dynamics of folding at high resolution using optical tweezers, measuring individual trajectories as nucleic acid hairpins passed through the high-energy transition states that dominate kinetics and define folding mechanisms. We observed brief but ubiquitous pauses in the transition states, with a dwell time distribution that matched microscopic theories of folding quantitatively. The sequence dependence suggested that pauses were dominated by microbarriers from nonnative conformations during the search by each nucleotide residue for the native base-pairing conformation. Furthermore, the pauses were position dependent, revealing subtle local variations in energy-landscape roughness and allowing the diffusion coefficient describing the microscopic dynamics within the barrier to be found without reconstructing the shape of the energy landscape. These results show how high-resolution measurements can elucidate key microscopic events during folding to test fundamental theories of folding.

Predicting and Simulating Mutational Effects on Protein Folding Kinetics.

Mutational perturbations of protein structures, i.e., phi-value analysis, are commonly employed to probe the extent of involvement of a particular residue in the rate-determining step(s) of folding. This generally involves the measurement of folding thermodynamic parameters and kinetic rate constants for the wild-type and mutant proteins. While computational approaches have been reasonably successful in understanding and predicting the effect of mutations on folding thermodynamics, it has been challenging to explore the same on kinetics due to confounding structural, energetic, and dynamic factors. Accordingly, the frequent observation of fractional phi-values (mean of ~0.3) has resisted a precise and consistent interpretation. Here, we describe how to construct, parameterize, and employ a simple one-dimensional free energy surface model that is grounded in the basic tenets of the energy landscape theory to predict and simulate the effect of mutations on folding kinetics. As a proof of principle, we simulate one-dimensional free energy profiles of 806 mutations from 24 different proteins employing just the experimental destabilization as input, reproduce the relative unfolding activation free energies with a correlation of 0.91, and show that the mean phi-value of 0.3 essentially corresponds to the extent of stabilization energy gained at the barrier top while folding.

Analysis of Molecular Dynamics Simulations of Protein Folding.

Unbiased molecular dynamics simulations of proteins can now capture spontaneous folding events. This provides a wealth of data reflecting information on folding mechanism, but raises the challenge of interpreting it in a meaningful way. Here, I describe how such simulations can be used to identify reactive states and reaction coordinates for describing folding, and how folding dynamics can be captured by projection onto those coordinates. Methods are described for quantifying the interactions important for defining the folding mechanism, and for comparison of simulations with experimental mechanistic probes, such as ϕ-values.

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme.

The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97-Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.

The A39G FF domain folds on a volcano-shaped free energy surface via separate pathways

Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect "invisible" minor states on the energy landscape of the A39G mutant FF domain that exhibited "two-state" folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ∼5 to 300 s-1 involving sparse states with populations as low as ∼1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ∼100 to ∼15,000 μs. A similar folding scheme is established for the wild-type domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES.

Chromosome Folding Promotes Intrachromosomal Aberrations under Radiation- and Nuclease-Induced DNA Breakage.

The long-standing question in radiation and cancer biology is how principles of chromosome organization impact the formation of chromosomal aberrations (CAs). To address this issue, we developed a physical modeling approach and analyzed high-throughput genomic data from chromosome conformation capture (Hi-C) and translocation sequencing (HTGTS) methods. Combining modeling of chromosome structure and of chromosomal aberrations induced by ionizing radiation (IR) and nuclease we made predictions which quantitatively correlated with key experimental findings in mouse chromosomes: chromosome contact maps, high frequency of cis-translocation breakpoints far outside of the site of nuclease-induced DNA double-strand breaks (DSBs), the distinct shape of breakpoint distribution in chromosomes with different 3D organizations. These correlations support the heteropolymer globule principle of chromosome organization in G1-arrested pro-B mouse cells. The joint analysis of Hi-C, HTGTS and physical modeling data offers mechanistic insight into how chromosome structure heterogeneity, globular folding and lesion dynamics drive IR-recurrent CAs. The results provide the biophysical and computational basis for the analysis of chromosome aberration landscape under IR and nuclease-induced DSBs.

Two energy barriers and a transient intermediate state determine the unfolding and folding dynamics of cold shock protein

Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques. Recently two-state property of Csp was confirmed by atomic force microscopy (AFM) through direct pulling measurement, while several long-lifetime intermediate states were found by force-clamp AFM. We systematically studied force-dependent folding and unfolding dynamics of Csp using magnetic tweezers with intrinsic constant force capability. Here we report that Csp mostly folds and unfolds with a single step over force range from 5 pN to 50 pN, and the unfolding rates show different force sensitivities at forces below and above ~8 pN, which determines a free energy landscape with two barriers and a transient intermediate state between them along one transition pathway. Our results provide a new insight on protein folding mechanism of two-state proteins.

Protein Folding Dynamics as Diffusion on a Free Energy Surface: Rate Equation Terms, Transition Paths, and Analysis of Single-Molecule Photon Trajectories.

The rates of protein (un)folding are often described as diffusion on the projection of a hyperdimensional energy landscape onto a few (ideally one) order parameters. Testing such an approximation by experiment requires resolving the reactive transition paths of individual molecules, which is now becoming feasible with advanced single-molecule spectroscopic techniques. This has also sparked the interest of theorists in better understanding reactive transition paths. Here we focus on these issues aiming to establish (i) practical guidelines for the mechanistic interpretation of transition path times (TPT) and (ii) methods to extract the free energy surface and protein dynamics from the maximum likelihood analysis of photon trajectories (MLA-PT). We represent the (un)folding rates as diffusion on a 1D free energy surface with the FRET efficiency as a reaction coordinate proxy. We then perform diffusive kinetic simulations on surfaces with two minima and a barrier, but with different shapes (curvatures, barrier height, and symmetry), coupled to stochastic simulations of photon emissions that reproduce current SM-FRET experiments. From the analysis of transition paths, we find that the TPT is inversely proportional to the barrier height (difference in free energy between minimum and barrier top) for any given surface shape, and that dividing the TPT into climb and descent segments provides key information about the barrier's symmetry. We also find that the original MLA-PT procedure used to determine the TPT from experiments underestimates its value, particularly for the cases with smaller barriers (e.g., fast folders), and we suggest a simple strategy to correct for this bias. Importantly, we also demonstrate that photon trajectories contain enough information to extract the 1D free energy surface's shape and dynamics (if TPT is >4-5-fold longer than the interphoton time) using the MLA-PT directly implemented with a diffusive free energy surface model. When dealing with real (unknown) experimental data, the comparison between the likelihoods of the free energy surface and discrete kinetic three-state models can be used to evaluate the statistical significance of the estimated free energy surface.

The trans-to-cis proline isomerization in E. coli Trx folding is accelerated by trans prolines

The conserved fold of thioredoxin (Trx)-like thiol/disulfide oxidoreductases contains an invariant cis-proline residue (P76 in Escherichia coli Trx) that is essential for Trx function and that is responsible for the folding rate-limiting step. E. coli Trx contains four additional prolines, which are all in the trans conformation in the native state. Notably, a recent study revealed that replacement of all four trans prolines in Trx by alanines (Trx variant Trx1P) further slowed the rate-limiting step 25-fold, indicating that one or several of the four trans prolines accelerate the trans-to-cis transition of P76 in Trx wild-type (wt). Here, we characterized the folding kinetics of Trx variants containing cisP76 and one or several of the natural trans prolines of Trx wt with NMR spectroscopy. First, we demonstrate that the isomerization reaction in Trx1P is a pure two-state transition between two distinct tertiary structures, in which all observed NMR resonances changes follow the same first-order kinetics. Moreover, we show that trans-P68 is the critical residue responsible for the faster folding of wt Trx relative to the single-proline (P76) variant Trx1P, as the two-proline variant Trx2P(P76P68) already folds seven times faster than Trx1P. trans-P34 also accelerates trans-to-cis isomerization of P76, albeit to a smaller extent. Overall, the results demonstrate that trans prolines can significantly modulate the kinetics of rate-limiting trans-to-cis proline isomerization in protein folding. Finally, we discuss possible mechanisms of acceleration and the potential significance of a protein-internal folding acceleration mechanism for Trx in a living cell.

Small Neutral Crowding Solute Effects on Protein Folding Thermodynamic Stability and Kinetics.

Molecular crowding is a ubiquitous phenomenon in biological systems, with significant consequences on protein folding and stability. Small compounds, such as the osmolyte trimethylamine N-oxide (TMAO), can also present similar effects. To analyze the effects arising from these solute-like molecules, we performed a series of crowded coarse-grained folding simulations. Two well-known proteins were chosen, CI2 and SH3, modeled by the alpha-carbon-structure-based model. In the simulations, the crowding molecules were represented by low-sized neutral atom beads in different concentrations. The results show that a low level of the volume fraction occupied by neutral agents can change protein stability and folding kinetics for the two systems. However, the kinetics were shown to be unaffected in their respective folding temperatures. The faster kinetics correlates with changes in the folding route for systems at the same temperature, where non-native contacts were shown to be relevant. The transition states of the two systems with and without crowders are similar. In the case of SH3, there are differences in the structuring of two strands, which may be associated with the increase in its folding rate, in addition to the destabilization of the denatured ensemble. The present study also detected a crossover in the thermodynamic stability behavior, previously observed experimentally and theoretically. As the temperature increases, crowders change from destabilizing to stabilizing agents.

Determining folding and binding properties of the C-terminal SH2 domain of SHP2.

SH2 domains are a class of protein–protein interaction modules with the function to recognize and bind sequences characterized by the presence of a phosphorylated tyrosine. SHP2 is a protein phosphatase involved in the Ras‐ERK1/2 signaling pathway that possess two SH2 domains, namely, N‐SH2 and C‐SH2, that mediate the interaction of SHP2 with various partners and determine the regulation of its catalytic activity. One of the main interactors of the SH2 domains of SHP2 is Gab2, a scaffolding protein with critical role in determining cell differentiation. Despite their key biological role and the importance of a correct native fold to ensure it, the mechanism of binding of SH2 domains with their ligands and the determinants of their stability have been poorly characterized. In this article, we present a comprehensive kinetic study of the folding of the C‐SH2 domain and the binding mechanism with a peptide mimicking a region of Gab2. Our data, obtained at different pH and ionic strength conditions and supported by site‐directed mutagenesis, highlight the role of electrostatic interactions in the early events of recognition. Interestingly, our results suggest a key role of a highly conserved histidine residue among SH2 family in the interaction with negative charges carried by the phosphotyrosine of Gab2. Moreover, the analysis of the equilibrium and kinetic folding data of C‐SH2 describes a complex mechanism implying a change in rate‐limiting step at high denaturant concentrations. Our data are discussed under the light of previous works on N‐SH2 domain of SHP2 and other SH2 domains.

The ubiquitous buried water in the beta-trefoil architecture contributes to the folding nucleus and ~20% of the folding enthalpy.

The beta-trefoil protein architecture is characterized by three repeating "trefoil" motifs related by rotational symmetry and postulated to have evolved via gene duplication and fusion events. Despite this apparent structural symmetry, the primary and secondary structural elements typically exhibit pronounced asymmetric features. A survey of this family of proteins has revealed that among the most conserved symmetric structural elements is a ubiquitous buried solvent which participates in a bridging H-bond with three different beta-strands in each of the trefoil motifs. A computational analysis reported that these waters are likely associated with a substantial enthalpic contribution to overall stability. In this report, a Pro mutation is used to disrupt one of the water H-bond interactions to a main chain amide, and the effects upon stability and folding kinetics are determined. Combined with Ala mutations, the separate effects upon side chain truncation and H-bond deletion are analyzed in terms of stability and folding kinetics. The results show that these buried waters act to assemble a central folding nucleus, and are responsible for ~20% of the overall favorable enthalpy of folding.

Elucidating molecular connetion between IAHSP onset and Alsin protein by means of Homology Modelling and Molecular Dynamics

The Infantile-onset Ascending Hereditary Spastic Paralysis (IAHSP) is an incurable rare neurodegerative disease related to a mutation-driven aberrant behaviour of the Alsin protein. The lack of information on Alsin atomic structure limits a complete understanding on pathology mechanisms. In this work, molecular modelling techniques have been applied to shed lights on Alsin folding dynamics and misfunction induced by aberrant mutations.

Structural localization of pathogenic mutations in the central nucleotide-binding domain (NBD) of nucleotide-binding oligomerization domain-2 (NOD2) protein and their inference in inflammatory disorders

The human NBD domain which is centrally located in the NOD2 protein displays an essential role in oligomerization and initiates the immune response via CARD-RIPK2 interaction. The mutations associated with the NBD domain have been largely implicated in inflammatory disorders such as Blau syndrome and sarcoidosis. This study aims to determine the structural and phenotypic effect of a lethal mutation that occurs in the NBD domain which has an axiomatic impact on protein dysfunction. Initially, the most deleterious missense mutations were screened through various in silico analysis. Out of 33 variants, I-Mutant 3.0, SIFT, PolyPhen 2, Align GVGD, PHD SNP and SNP&GO have statistically identified 5 variants (R42W, D90E, E91K, G189D & W198L) as less stable, deleterious and damaging. Our predicted models have paved the way to understand the various structural properties such as physiochemical, secondary structural arrangements and stabilizing residues in folding associated with the native and mutant NBD domain especially of the functionally important regions. From the aforementioned results, R42W and G189D were found to be the more predominant among the mutants. Precisely, through molecular simulation, we have strongly justified the significant conformational disruption of R42W and G189D through the stabilization factors, folding and essential dynamics. Conclusively, these regions (α341-44, α13185-191 and β6133-143β7) seem to adopt such structures that are not conducive to wild-type-like functionality. Our prediction and validation of lethal mutations based on structural stability may be useful for conducting experimental studies in detail to uncover the protein deregulation leading to inflammatory disorders.

Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.