Unfolding Transition Research Articles

Autoregressive HMM resolves biomolecular transitions from passive optical tweezer force measurements

Optical tweezer (OT) single molecule force spectroscopy is a powerful method to map out the energy landscape of biological complexes and has found increasing applications in academic and pharmaceutical research. The dominant method to extract molecular conformation transitions from the thermal diffusion-broadened trajectories of the microscopic OT probes attached to the single molecule of interest is through hidden Markov models (HMM). In standard applications, the HMMs assume a white noise spectrum of the probes superimposed onto the molecular signal.Here, we demonstrate, through theoretical derivation, computer modeling and experimental measurements that this standard white noise HMM (wnHMM) misses key features of real OT data. The deviation is most pronounced at higher frequencies because the white noise model does not account for the over-damped nature of particle diffusion in an OT harmonic potential in aqueous environments. To address this, we derive how to incorporate autoregression (ar) between consecutive data points into an HMM, and demonstrate through modeling and experiment that such an arHMM captures real OT data behavior across all frequency ranges. Through analysis of real OT data we recorded on a single DNA hairpin undergoing folding and unfolding transitions, we show that the wnHMM extracts lifetimes that are at least a factor of 2 faster and less consistent than the arHMM results which match expectations and prior measurements. Overall, our work suggests that arHMM should be the default model choice for analysis OT single molecule transitions and that its use will improve the fidelity and accuracy of single molecule force spectroscopy measurements.

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.

Survey of Chemical Unfolding Complexity as a Unique Stability Assessment Assay for Monoclonal Antibodies

Seventy-two intentionally sequence-diverse antibody variable regions were selected, expressed as IgG1 antibodies, and evaluated by chemical unfolding to survey the complexities of denaturant induced unfolding behavior. A two-transition fit well described the curves and uncovered a wide range of sensitivities to denaturant. Four general types of unfolding curves were observed: balanced traces (each transition responsible for half of the total unfolding transition), low-unfolding traces (first transition is a majority of the unfolding curve), high-unfolding traces (the second transition is the majority of the unfolding curve), and coincident traces (the two transitions are found close to each other, approximating a single transition).The complexity of the data from this survey indicates that focusing on the first inflection point or fitting a single transition model is likely an over-simplistic method for measuring stability by the chemical unfolding assay. Additionally, other conformational assays, such as thermal and low pH unfolding, showed no correlation with the chemical unfolding results, indicating that each of these assays provide alternate information on the different pathways of antibody conformational stability. These results provide a basis for beginning to better connect unfolding behavior to other physical phenotypic behaviors and production process behaviors.

Materializing responsible futures: An interpretative phenomenological analysis of circular design experiences in construction

Reimagining design as a transformative practice for realizing a circular built environment is both urgent and important. Many of today's resource problems can be traced back to the way constructions are being designed. The adoption of circular design practices may alleviate these problems. Most previous research has either mapped the boundaries of contemporary circular design practices or pushed those boundaries with new interventions. The lived experiences of designers are, however, often overlooked. Little remains known about what it is like to be engaged in and how to ‘live through’ circular design. This study therefore seeks to understand the practice from the perspective of designers themselves. Through applying an interpretative phenomenological analysis to unstructured interview data collected from ten frontrunning Dutch designers, it explores both the what and how of circular design. Four emergent themes were found that illuminate the experience itself. Circular design is, accordingly, interpreted as a practice which: proclaims responsibility towards the Earth, materializes future-oriented solutions, deals with a multi-headed monster, and involves orchestrating a design ecosystem. These themes are illustrated with narrative accounts of designers' actual experiences. The rich, in-depth insights offer ample learning opportunities to better understand and facilitate unfolding circularity transitions. Circular design is, as such, theorized as a vital practice that can shape the built environment through materializing responsible futures.

Stable monomers in the ancestral sequence reconstruction of the last opisthokont common ancestor of dimeric triosephosphate isomerase.

Function and structure are strongly coupled in obligated oligomers such as Triosephosphate isomerase (TIM). In animals and fungi, TIM monomers are inactive and unstable. Previously, we used ancestral sequence reconstruction to study TIM evolution and found that before these lineages diverged, the last opisthokonta common ancestor of TIM (LOCATIM) was an obligated oligomer that resembles those of extant TIMs. Notably, calorimetric evidence indicated that ancestral TIM monomers are more structured than extant ones. To further increase confidence about the function, structure, and stability of the LOCATIM, in this work, we applied two different inference methodologies and the worst plausible case scenario for both of them, to infer four sequences of this ancestor and test the robustness of their physicochemical properties. The extensive biophysical characterization of the four reconstructed sequences of LOCATIM showed very similar hydrodynamic and spectroscopic properties, as well as ligand-binding energetics and catalytic parameters. Their 3D structures were also conserved. Although differences were observed in melting temperature, all LOCATIMs showed reversible urea-induced unfolding transitions, and for those that reached equilibrium, high conformational stability was estimated (ΔGTot = 40.6-46.2 kcal/mol). The stability of the inactive monomeric intermediates was also high (ΔGunf = 12.6-18.4 kcal/mol), resembling some protozoan TIMs rather than the unstable monomer observed in extant opisthokonts. A comparative analysis of the 3D structure of ancestral and extant TIMs shows a correlation between the higher stability of the ancestral monomers with the presence of several hydrogen bonds located in the "bottom" part of the barrel.

Targeting the protein folding transition state by mutation: Large scale (un)folding rate accelerations without altering native stability.

Proteins are constantly undergoing folding and unfolding transitions, with rates that determine their homeostasis in vivo and modulate their biological function. The ability to optimize these rates without affecting overall native stability is hence highly desirable for protein engineering and design. The great challenge is, however, that mutations generally affect folding and unfolding rates with inversely complementary fractions of the net free energy change they inflict on the native state. Here we address this challenge by targeting the folding transition state (FTS) of chymotrypsin inhibitor 2 (CI2), a very slow and stable two-state folding protein with an FTS known to be refractory to change by mutation. We first discovered that the CI2's FTS is energetically taxed by the desolvation of several, highly conserved, charges that form a buried salt bridge network in the native structure. Based on these findings, we designed a CI2 variant that bears just four mutations and aims to selectively stabilize the FTS. This variant has >250-fold faster rates in both directions and hence identical native stability, demonstrating the success of our FTS-centric design strategy. With an optimized FTS, CI2 also becomes 250-fold more sensitive to proteolytic degradation by its natural substrate chymotrypsin, and completely loses its activity as inhibitor. These results indicate that CI2 has been selected through evolution to have a very unstable FTS in order to attain the kinetic stability needed to effectively function as protease inhibitor. Moreover, the CI2 case showcases that protein (un)folding rates can critically pivot around a few key residues-interactions, which can strongly modify the general effects of known structural factors such as domain size and fold topology. From a practical standpoint, our results suggest that future efforts should perhaps focus on identifying such critical residues-interactions in proteins as best strategy to significantly improve our ability to predict and engineer protein (un)folding rates.

Mechanical Stability and Unfolding Pathways of Parallel Tetrameric G-Quadruplexes Probed by Pulling Simulations.

Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects remain elusive, despite being important for understanding structure formation and biological functions of GQs. Pulling experiments are a common tool for acquiring insights into the folding landscape of GQs. Herein, we applied a computational pulling strategy─steered molecular dynamics (SMD) simulations─in combination with standard molecular dynamics (MD) simulations to explore the unfolding landscapes of tetrameric parallel GQs. We identified anisotropic properties of elastic conformational changes, unfolding transitions, and GQ mechanical stabilities. Using a special set of structural parameters, we found that the vertical component of pulling force (perpendicular to the average G-quartet plane) plays a significant role in disrupting GQ structures and weakening their mechanical stabilities. We demonstrated that the magnitude of the vertical force component depends on the pulling anchor positions and the number of G-quartets. Typical unfolding transitions for tetrameric parallel GQs involve base unzipping, opening of the G-stem, strand slippage, and rotation to cross-like structures. The unzipping was detected as the first and dominant unfolding event, and it usually started at the 3'-end. Furthermore, results from both SMD and standard MD simulations indicate that partial spiral conformations serve as a transient ensemble during the (un)folding of GQs.

Optimized finite-time work protocols for the Higgs RNA model with external force.

The Higgs RNA model with an added term for a coupling to an external force is studied in regard to finite-time force-driving protocols with a minimal-work requirement. In this paper, RNA sequences which at low temperature exhibit hairpins are considered, which are often cited as typical template systems in stochastic thermodynamics. The optimized work protocols for this glassy many-particle system are determined numerically using the parallel tempering method. The protocols show distinct jumps at the beginning and end, which have been observed for single-particle systems and are proven to be optimal in the fast protocol limit generally. Optimality seems to be achieved by staying close to the equilibrium unfolding transition point, in agreement with experimental and theoretical observations. The change of work distributions, compared to those resulting from a naive linear driving protocol, are discussed generally and in terms of free energy estimation as well as the effect of optimized protocols on rare work process starting conditions.

Challenges in accelerating net-zero transitions: insights from transport electrification in Germany and California

Addressing the climate crisis necessitates accelerating transitions towards climate-neutral systems of production and consumption, with electrification emerging as a crucial decarbonisation strategy. The acceleration of such net-zero transitions across multiple systems faces significant resistance and contestation. In this paper, we propose an extended list of challenges unique to the acceleration phase of socio-technical transitions: we introduce ‘expansion and contestation’, ‘justice’, and ‘international dynamics’ as additional challenge types to complement the already acknowledged challenge types of ‘whole systems change’, ‘interaction between multiple systems’, ‘decline and resistance’, ‘consumers and social practices’, and ‘governance’. We apply this extended analytical framework to the electrification of private-passenger vehicles and investigate the unfolding transition to e-mobility with evidence from 35 expert interviews in Germany and California. We uncover over 50 real-world challenges associated with these net-zero transitions at the beginning of the acceleration phase. Most challenges fall within the categories of ‘expansion and contestation’ and ‘governance’. While Germany and California share many real-world challenges, we also find significant variation between both jurisdictions, which we attribute to differences in their automotive incumbency, transition governance approaches, and institutional contexts. We discuss implications for future research, arguing for greater attention to the dual politics of acceleration during net-zero transitions: political conflict not only centres around the decline of old industries and future losses, but also around the expansion of the new system and associated future gains.

Human apo-metallothionein 1a is not a random coil: Evidence from guanidinium chloride, high temperature, and acidic pH unfolding studies

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.

The Biochemical Impact of Extracting an Embedded Adenylate Kinase Domain Using Circular Permutation.

Adenylate kinases (AKs) have evolved AMP-binding and lid domains that are encoded as continuous polypeptides embedded at different locations within the discontinuous polypeptide encoding the core domain. A prior study showed that AK homologues of different stabilities consistently retain cellular activity following circular permutation that splits a region with high energetic frustration within the AMP-binding domain into discontinuous fragments. Herein, we show that mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologues at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30 °C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than those of native AKs, and they exhibited multiphasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK in differing manners, and mass spectrometry suggested that the new termini within the AMP-binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes.

Exploring the free energy landscape of proteins using magnetic tweezers.

Proteins fold to their native states by searching through the free energy landscapes. As single-domain proteins are the basic building block of multiple-domain proteins or protein complexes composed of subunits, the free energy landscapes of single-domain proteins are of critical importance to understand the folding and unfolding processes of proteins. To explore the free energy landscapes of proteins over large conformational space, the stability of native structure is perturbed by biochemical or mechanical means, and the conformational transition process is measured. In single molecular manipulation experiments, stretching force is applied to proteins, and the folding and unfolding transitions are recorded by the extension time course. Due to the broad force range and long-time stability of magnetic tweezers, the free energy landscape over large conformational space can be obtained. In this article, we describe the magnetic tweezers instrument design, protein construct design and preparation, fluid chamber preparation, common-used measuring protocols including force-ramp and force-jump measurements, and data analysis methods to construct the free energy landscape. Single-domain cold shock protein is introduced as an example to build its free energy landscape by magnetic tweezers measurements.

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II.

Variable temperature electrospray mass spectrometry is useful for multiplexed measurements of the thermal stabilities of biomolecules, but the ionization process can be disrupted by aggregation-prone proteins/complexes that have irreversible unfolding transitions. Resistively heating solutions containing a mixture of bovine carbonic anhydrase II (BCAII), a CO2 fixing enzyme involved in many biochemical pathways, and cytochrome c leads to complete loss of carbonic anhydrase signal and a significant reduction in cytochrome c signal above ∼72 °C due to aggregation. In contrast, when the tips of borosilicate glass nanoelectrospray emitters are heated with a laser, complete thermal denaturation curves for both proteins are obtained in <1 minute. The simultaneous measurements of the melting temperature of BCAII and BCAII bound to bicarbonate reveal that the bicarbonate stabilizes the folded form of this protein by ∼6.4 °C. Moreover, the temperature dependences of different bicarbonate loss pathways are obtained. Although protein analytes are directly heated by the laser for only 140 ms, heat conduction further up the emitter leads to a total analyte heating time of ∼41 s. Pulsed laser heating experiments could reduce this time to ∼0.5 s for protein aggregation that occurs on a faster time scale. Laser heating provides a powerful method for studying the detailed mechanisms of cofactor/ligand loss with increasing temperature and promises a new tool for studying the effect of ligands, drugs, growth conditions, buffer additives, or other treatments on the stabilities of aggregation-prone biomolecules.

Biophysical characterization of human-cell-expressed, full-length κI O18/O8, AL-09, λ6a, and Wil immunoglobulin light chains

Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.

The NRRP and the Italian energy transition. Interest groups in implementation, between structural power, insiderness and new coalitions

ABSTRACT Italian energy policy has traditionally been significantly influenced by large state-owned companies, commonly referred to as national champions, as well as by fossil fuel interests. The impact of the energy transition on this situation has been limited, as the adoption of renewables in Italy has not been accompanied by the emergence of a robust green coalition. The National Recovery and Resilience Plan provided an opportunity to address this situation. Through process tracing and interviews with key policy actors, this article examines the implementation of energy transition measures outlined in the plan during the recent administrations of Draghi and Meloni. The analysis underscores the changing role of relevant interest groups and business actors in this sector. It reveals that the structural power of national champions and the influential position of core insiders have not diminished, particularly following adjustments to the recovery plan prompted by the Russia-Ukraine war. Nonetheless, the article notes that green outsiders have, through coalition dynamics, increased their involvement in policy implementation. Additionally, it emphasizes that the unfolding energy transition is driving a reconfiguration of interests that transcends the traditional divide between fossil fuels and renewable energy.

Mechanistic insights into the improving effects of germination on physicochemical properties and antioxidant activity of protein isolate derived from black and white sesame.

Germination is a natural green technology to improve the nutritional and techno-functional quality of plant-based proteins. In this study, the mechanism of improving the functional and antioxidant properties of black and white sesame protein isolates (SPI) through germination process was investigated. Results showed that the surface hydrophobicity and sulfhydryl content increased significantly after germination, which were supported by multispectral analysis suggesting the exposed and unfolded conformational transition of germinated SPI. Moreover, the increased particle size was observed by microscopy analysis and reducing electrophoresis, which indicated that depolymerized protein molecules were rearranged to form protein aggregates during germination. The structural modification induced by germination contributed to the superior solubility (increased to 3.15-fold and 2.36-fold at pH 8 for black and white SPI, respectively), foaming capacity (increased to 3.99-fold and 1.69-fold, respectively), emulsifying ability (increased to 2.84-fold and 2.71-fold, respectively), and diverse chemical antioxidant activities (increased up to 5.60-fold) of SPI in both varieties. This was the first comprehensive study to investigate germination as a promising technology for obtaining high-quality SPI.

Detection of Hofmeister effects at a single-molecule level

Metal cations are essential for the function of nucleic acids. Given their efficiency for DNA overwinding and the DNA hairpin folding/unfolding process coupled with DNA twist, we systematically investigated the effects of cation identities and increasing concentrations of NaCl, KCl, and LiCl from 50 mM to 300 mM on the folding/unfolding process by manipulating a DNA hairpin construct with magnetic tweezers. The critical forces and free energy of the unfolding transition of the DNA hairpin at a fixed concentration of KCl, NaCl, and LiCl followed the same ranking as , and are reversed with the unfolding rate ranking of {}\ ext{Na}^{+}>{}\ ext{Li}^{+}$ ?> . This highlights that the addition of Li+ in solution obviously enhances the stability of the DNA hairpin when compared to Na+, and K+, and their efficiency on the stability of the DNA hairpin was ranked as .

Dark nanodiscs for evaluating membrane protein thermostability by differential scanning fluorimetry

Measuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed nonfluorescent membrane scaffold protein to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the “dark” nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs loaded with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) lipids show a complex biphasic thermal unfolding pattern with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5°C and 77.5°C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a DMPC and DMPG nanodisc. To extend the utility of this method, we evaluate and compare the thermostability of DsbB in different lipid environments. We introduce this method as a new tool that can be used to understand how compositionally and biophysically complex lipid environments drive membrane protein stability.

Unfolding low-carbon reorientation in a declining industry: A contextual analysis of changing company strategies in UK oil refining (1990–2023)

The oil refining decarbonisation literature makes important analyses of technical and economic dimensions of low-carbon transition pathways but pays less attention to the contexts and strategic considerations that shape the practical implementation of low-carbon technologies by refineries, including investment decisions. To address this understudied issue, this paper makes a longitudinal analysis of changing external pressures and company response strategies in the UK's refining industry from 1990, when climate change rose on socio-political agendas, to 2023. We apply the Triple Embeddedness Framework, a five-phase model of industry reorientation, to analyse the unfolding low-carbon transition process over five successive periods (1990–2000, 2000–2008, 2008–2015, 2015–2019, 2019–2023). Using information from policy documents, reports by industry associations, company annual reports (including financial reporting), academic and practitioner publications, and 24 interviews with academics and industry experts, our analysis finds that the UK's refining industry moved from inaction and incremental change in the first period, to some exploration of alternatives in the second period, and back to incremental change in the third period (after the 2008 financial crisis). In the fourth period, refineries more seriously started to explore low-carbon technologies, which they began to deploy in the fifth period, notably carbon capture and storage (CCS) and low-carbon hydrogen. The paper analyses and explains this non-linear reorientation trajectory and elucidates why refineries have embarked on significant low-carbon reorientation despite long-term decline in the wider industry.

Unfolding sustainability transitions in food systems: Insights from UK and French trajectories.

While the negative environmental, social and health impacts of the current food system have been acknowledged and evidenced for several decades, the recent and current transformations in food systems at diverse scales are not yet addressing the many inter-related stakes at play. Due to the much wider set of interactions in this consumption-production system, new conceptual tools are required for understanding and assessing sustainability transitions and what prevents them. The article will draw on the cases of France and the UK to examine these countries' national food systems' historical trajectories and suggest a periodization of these in order to reveal common characteristics and differences. This will show that despite common major trends and common transition or inertia mechanisms, pathways differ, especially from the 1990s, due to different configurations of power relationships between the state, economic actors and civil society in a context of an increasing competition between sustainability narratives that leads to an increasing fragmentation in food systems. It will lead us to join the recent progress in the sustainability transitions' community towards a shift in the analysis from a focus on niches' trajectories and effects to a deeper focus on power configurations and competing narratives, as well as to suggest a larger inclusion of socio-ecological and spatial dimensions.