Unfolding Transition Research Articles

Polyols, increasing global stability of cytochrome c, destabilize the thermal unfolding intermediate

Studies on the intermediate states of proteins provide essential information on folding pathway and energy landscape of proteins. Osmolytes, known to alter the stability of proteins, might also affect the structure and energy states of folding intermediates. This was examined using cytochrome c (Cyt) as a model protein which forms a spectroscopically detectable intermediate during thermal denaturation transition. Most of the secondary structure and the native heme-ligation were intact in the intermediate state of the protein. Denaturants, urea and guanidinium hydrochloride, and ionic salt destabilizes the intermediate and drive the protein to follow two-state transition. The effect of polyol class of osmolytes, glycol, glycerol, erythritol, xylitol and sorbitol (with OH-groups two to six), on the intermediate was studied using Soret absorbance and far-UV circular dichroism. With the increasing concentration of any of the polyols, the transition-midpoint temperature (Tm ) and the enthalpy change (ΔH) for native to intermediate transition were decreased. This indicated that the intermediate was destabilized by the polyols. However, the polyols increased the overall stability of the protein by increasing Tm and ΔH for intermediate to unfolded transition, except for glycol which destabilized the protein. These results show that the polyols could alter the energy state of the intermediate, and the effect of lower and higher polyols might be different on the stability and folding pathway of the protein. Communicated by Ramaswamy H. Sarma

Size-Selective VAILase Proteolysis Provides Dynamic Insights into Protein Structures.

Monitoring the dynamic alterations of protein structures within an aqueous solution remains enormously challenging. In this study, we describe a size-selective VAILase proteolysis (SVP)-mass spectrometry (MS) strategy to probe the protein structure changes without strict control of the proteolysis kinetics. The unique conformation selectivity of SVP depends on the uniform nano-sized entrance pores of the VAILase hexameric cage as well as the six inherent molecular rulers in the VAILase-substrate recognition and cleavage. The dynamic insights into subtle conformation alterations of both myoglobin unfolding transition and Aurora kinase A-inhibitor binding are successfully captured using the SVP strategy, which matches well with the results in the molecular dynamics simulation. Our work provides a new paradigm of size-selective native proteolysis for exploring the aqueous protein structure-function relationships.

The effects of intramolecular and intermolecular electrostatic repulsions on the stability and aggregation of NISTmAb revealed by HDX-MS, DSC, and nanoDSF.

The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX-MS), differential scanning calorimetry (DSC), and nano-differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX-MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the CH 2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry-Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI.

Hype cycles during socio-technical transitions: The dynamics of collective expectations about renewable energy in Germany

The literature on socio-technical change describes the widely acknowledged, important roles of collectively shared expectations in shaping the trajectories of novel technologies. However, in previous studies on these technological expectations, researchers have mainly emphasised early innovation stages, paying little attention to technologies that have already begun to challenge and transform the established socio-technical system. In this study, we use the example of the German energy transition to address this gap. By conducting a content analysis of nearly 12,000 newspaper articles, we examined the nature and dynamics of expectations that were circulating in Germany's wider public from 1992 to 2017 regarding wind power, solar photovoltaics, and biogas. Our findings reveal patterns of hype and disillusionment for all three technologies and illustrate the important roles of frame expectations in shaping these hype cycles. Furthermore, the findings shed new light on processes that contribute to the emergence and dynamics of widely shared expectations and indicate that the associated hype patterns are manifestations of unfolding transition dynamics rather than the result of frustrated expectations. In theoretical terms, we integrated insights from (transition) research on discourse and framing into the sociology of expectations, initiating a promising dialogue between scholars in these two fields.

The merits of becoming smart: How Flemish and Dutch energy communities mobilise digital technology to enhance their agency in the energy transition

Recent developments in Information and Communication Technology (ICT) and ongoing digitalisation processes play key roles in the energy transition. It is often argued that digital technology has the potential to empower citizens and communities and thereby contribute not only to a more sustainable but also to a more democratic and fairer energy system. It is however unclear how the increasing number of frontrunner energy communities that enter the field of smart grids can benefit from these novel ICT solutions and meaningfully contribute to the sustainable energy transition. This paper explores how energy communities can mobilise ICT to enhance their agency in the energy transition. As part of a holistic multiple-case study approach, two energy communities were closely followed over a period of three years. Data were collected during project meetings and through semi-structured interviews. This paper observes that energy communities can mobilise ICT to change the way technology operates, strengthen collaboration to increase collective agency and, support their efforts in creating, disrupting, or maintaining institutions. The studied energy communities adopted a ‘fit and transform’ strategy in which they mobilised ICT to fit in the incumbent energy system in the short term, while aiming for transformation in the long term. ICT however also creates new challenges in the form of interoperability issues. This paper calls for more attention on the role of ICT when studying agency in unfolding sustainability transitions, especially in fields in which digital technology is believed to play a major role in the transformation.

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy left( {Delta {text{G}}_{{{text{NU}}}}^{{{text{H}}_{2} {text{O}}}} } right) of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production.

Statistical Learning from Single-Molecule Experiments: Support Vector Machines and Expectation-Maximization Approaches to Understanding Protein Unfolding Data.

Single-molecule force spectroscopy has become a powerful tool for the exploration of dynamic processes that involve proteins; yet, meaningful interpretation of the experimental data remains challenging. Owing to low signal-to-noise ratio, experimental force-extension spectra contain force signals due to nonspecific interactions, tip or substrate detachment, and protein desorption. Unravelling of complex protein structures results in the unfolding transitions of different types. Here, we test the performance of Support Vector Machines (SVM) and Expectation Maximization (EM) approaches in statistical learning from dynamic force experiments. When the output from molecular modeling in silico (or other studies) is used as a training set, SVM and EM can be applied to understand the unfolding force data. The maximal margin or maximum likelihood classifier can be used to separate experimental test observations into the unfolding transitions of different types, and EM optimization can then be utilized to resolve the statistics of unfolding forces: weights, average forces, and standard deviations. We designed an EM-based approach, which can be directly applied to the experimental data without data classification and division into training and test observations. This approach performs well even when the sample size is small and when the unfolding transitions are characterized by overlapping force ranges.

Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

Deleterious effects of strategic, profit-seeking energy storage operation on electric power system costs

Energy storage will play a key role in the unfolding energy transition, but current market design and the modeling efforts that inform discussions surrounding its role broadly assume that these systems will not be deployed in ways deleterious to the objectives of the wider power grid. This is myopic: there are plausible and strategic modes of storage operation that clash with wider grid objectives while maximizing profits for storage system owners. Here we compare three modes: one that is commonly modeled and is broadly beneficial to the power system—peak shaving—with two plausible but deleterious ones—load leveraging and ramp augmenting—and show how the latter two increase system costs and help generators earn substantial profits even when they bid at marginal cost. Our work adds to a growing literature about the unintended consequences of energy storage on the power system and elaborates the essential role of policy makers and regulators in mitigating these effects.

Stability of the Retinoid X Receptor-α Homodimer in the Presence and Absence of Rexinoid and Coactivator Peptide.

Differential scanning calorimetry and differential scanning fluorimetry were used to measure the thermal stability of human retinoid X receptor-α ligand binding domain (RXRα LBD) homodimer in the absence or presence of rexinoid and coactivator peptide, GRIP-1. The apo-RXRα LBD homodimer displayed a single thermal unfolding transition with a Tm of 58.7 °C and an unfolding enthalpy (ΔH) of 673 kJ/mol (12.5 J/g), much lower than average value (35 J/g) of small globular proteins. Using a heat capacity change (ΔCp) of 15 kJ/(mol K) determined by measurements at different pH values, the free energy of unfolding (ΔG) of the native state was 33 kJ/mol at 37 °C. Rexinoid binding to the apo-homodimer increased Tm by 5 to 9 °C and increased the ΔG of the native homodimer by 12 to 20 kJ/mol at 37 °C, consistent with the nanomolar dissociation constant (Kd) of the rexinoids. GRIP-1 binding to holo-homodimers containing rexinoid resulted in additional increases in ΔG of 14 kJ/mol, a value that was the same for all three rexinoids. Binding of rexinoid and GRIP-1 resulted in a combined 50% increase in unfolding enthalpy, consistent with reduced structural fluidity and more compact folding observed in other published structural studies. The complexes of UAB110 and UAB111 are each more stable than the UAB30 complex by 8 kJ/mol due to enhanced hydrophobic interactions in the binding pocket because of their larger end groups. This increase in thermodynamic stability positively correlates with their improved RXR activation potency. Thermodynamic measurements are thus valuable in predicting agonist potency.

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1.

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway.

Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations

Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to μm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α3D using atomic force microscopy (AFM)-based force spectroscopy. α3D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α3D's configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy's ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α3D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam-modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 kBT) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Characterization of Camkiialpha Holoenzyme Stability

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation. CaMKII comprises 2% of the total protein in dendritic spines, where it plays important signaling roles allowed for by its unique structure. Each CaMKII subunit is comprised of a kinase domain, regulatory segment, linker region, and hub domain that is responsible for oligomerizing the subunits. Understanding the hub domain and its dynamics (i.e., how it assembles and falls apart) is crucial, and we have pursued experiments to interrogate holoenzyme stability using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability, which is significantly stabilized by regulatory segment binding. The hub domain alone is extremely stable, whereas the holoenzyme structure has multiple unfolding transitions ranging from ∼60°C to 100°C. Using MP, we compared the masses CaMKIIα splice variants with different variable linker regions and determined that the holoenzymes are less stable) compared with the hub domain alone. These results indicate that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. Insight into the dynamics of the holoenzyme gives us a broader understanding of CaMKII activation-triggered subunit exchange. Active CaMKII exchanges subunits with neighboring CaMKIIs and spreads its activation state. This might have a significant role in memory formation where it remains unclear how memories are stored for long periods of time. We interrogated CaMKII subunit exchange using FRET and single-molecule microscopy. Our results indicate that stability correlates with subunit exchange rate. Some instability may be required to initiate this dynamic process. These data provide important insight to holoenzyme biophysics and its role in CaMKII biology.

Role of Salt Bridges in Ubiquitin in Modulating Thermodynamic, Kinetic and Mechanical Stabilities

The native-state structure and folding pathways of a protein are encoded in its amino acid sequence. Ubiquitin a post translational modifier is mainly noted for its function in intracellular protein degradation has two salt bridges: one relatively exposed (SB1:K11-E34) and the other buried (SB2:K27-D52). Here we investigate the role of charged residues and hydrophobic interactions in protein folding by mutating salt-bridge residues in ubiquitin with hydrophobic residues. The thermodynamic stability of SB1 null variant is increased whereas the stability of the SB2 null variant is slightly reduced. We prepared a double salt bridge (DB) null variant and its thermodynamic stability is an additive effect of the individual salt bridges. Kinetic experiments reveal that that all the salt-bridge null variants fold through a more stable intermediate with relatively faster folding rates. The SB2 null variant has a highly stabilized unfolding transition state (TS) and a slightly destabilized native state, leading to its kinetic instability, whereas the kinetic stability of SB1 null variant is not compromised as its TS and native state are stabilized to a similar extent. The TS stabilization is also additive for the DB null variant having the most stabilized TS and high kinetic instability. Our results underscore the importance of kinetic stability in optimizing protein energy landscape. Our study establishes the fact that the TSs can be stabilized by hydrophobic residues in the place of buried charged residues and the effect can be even reverse of that observed for the native state. It further highlights the role of charged residues in the protein interior in modulating the thermodynamic and kinetic stabilities. In future we are going to investigate the role of salt bridges in tuning the mechanical properties of ubiquitin.

Unfolding of the chromatin fiber driven by overexpression of noninteracting bridging factors

Nuclear molecules control the functional properties of the chromatin fiber by shaping its morphological properties. The biophysical mechanisms controlling how bridging molecules compactify chromatin are a matter of debate. On the one side, bridging molecules could cross-link faraway sites and fold the fiber through the formation of loops. Interacting bridging molecules could also mediate long-range attractions by first tagging different locations of the fiber and then undergoing microphase separation. Using a coarse-grained model and Monte Carlo simulations, we study the conditions leading to compact configurations both for interacting and noninteracting bridging molecules. In the second case, we report on an unfolding transition at high densities of the bridging molecules. We clarify how this transition, which disappears for interacting bridging molecules, is universal and controlled by entropic terms. In general, chains are more compact in the case of interacting bridging molecules because interactions are not valence limited. However, this result is conditional on the ability of our simulation methodology to relax the system toward its ground state. In particular, we clarify how, unless using reaction dynamics that change the length of a loop in a single step, the system is prone to remain trapped in metastable, compact configurations featuring long loops.

Machine learning reveals hidden stability code in protein native fluorescence

Conformational stability of a protein is usually obtained by spectroscopically measuring the unfolding melting temperature. However, optical spectra under native conditions are considered to contain too little resolution to probe protein stability. Here, we have built and trained a neural network model to take the temperature-dependence of intrinsic fluorescence emission under native-only conditions as inputs, and then predict the spectra at the unfolding transition and denatured state. Application to a therapeutic antibody fragment demonstrates that thermal transitions obtained from the predicted spectra correlate highly with those measured experimentally. Crucially, this work reveals that the temperature-dependence of native fluorescence spectra contains a high-degree of previously hidden information relating native ensemble features to stability. This could lead to rapid screening of therapeutic protein variants and formulations based on spectroscopic measurements under non-denaturing temperatures only.

Hierarchical supramolecular assembly of a single peptoid polymer into a planar nanobrush with two distinct molecular packing motifs

Hierarchical nanomaterials have received increasing interest for many applications. Here, we report a facile programmable strategy based on an embedded segmental crystallinity design to prepare unprecedented supramolecular planar nanobrush-like structures composed of two distinct molecular packing motifs, by the self-assembly of one particular diblock copolymer poly(ethylene glycol)-block-poly(N-octylglycine) in a one-pot preparation. We demonstrate that the superstructures result from the temperature-controlled hierarchical self-assembly of preformed spherical micelles by optimizing the crystallization-solvophobicity balance. Particularly remarkable is that these micelles first assemble into linear arrays at elevated temperatures, which, upon cooling, subsequently template further lateral, crystallization-driven assembly in a living manner. Addition of the diblock copolymer chains to the growing nanostructure occurs via a loosely organized micellar intermediate state, which undergoes an unfolding transition to the final crystalline state in the nanobrush. This assembly mechanism is distinct from previous crystallization-driven approaches which occur via unimer addition, and is more akin to protein crystallization. Interestingly, nanobrush formation is conserved over a variety of preparation pathways. The precise control ability over the superstructure, combined with the excellent biocompatibility of polypeptoids, offers great potential for nanomaterials inaccessible previously for a broad range of advanced applications.

Conserved patterns and interactions in the unfolding transition state across SH3 domain structural homologues.

Proteins with similar structures are generally assumed to arise from similar sequences. However, there are more cases than not where this is not true. The dogma is that sequence determines structure; how, then, can very different sequences fold to the same structure? Here, we employ high temperature unfolding simulations to probe the pathways and specific interactions that direct the folding and unfolding of the SH3 domain. The SH3 metafold in the Dynameomics Database consists of 753 proteins with the same structure, but varied sequences and functions. To investigate the relationship between sequence and structure, we selected 17 targets from the SH3 metafold with high sequence variability. Six unfolding simulations were performed for each target, transition states were identified, revealing two general folding/unfolding pathways at the transition state. Transition states were also expressed as mathematical graphs of connected chemical nodes, and it was found that three positions within the structure, independent of sequence, were consistently more connected within the graph than any other nearby positions in the sequence. These positions represent a hub connecting different portions of the structure. Multiple sequence alignment and covariation analyses also revealed certain positions that were more conserved due to packing constraints and stabilizing long-range contacts. This study demonstrates that members of the SH3 domain with different sequences can unfold through two main pathways, but certain characteristics are conserved regardless of the sequence or unfolding pathway. While sequence determines structure, we show that disparate sequences can provide similar interactions that influence folding and lead to similar structures.

Biophysical characterization of the structural stability of Helix lucorum hemocyanin

The structural stability of the hеmocyanin purified from the hеmolymph of gаrden snаils Helix lucorum (HlH) was investigated by means of far-UV circular dichroism (CD), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). For the first time, TEM analyses showed the presence of tubular polymers in hemocyanins after three-day dialysis against a stabilizing buffer containing high concentrations of Ca2+ and Mg2+ ions (100 mmol L −1). The conformational stability study of native HlH by means of CD in a wide pH range (2.5-11.5) defined the pH stability region of HlH at pH 6.5 − 8.0. DSC analyses demonstrated the thermal stability of this hemocyanin. One transition, with an apparent transition tеmperature (T m) at 82.3 °C, was detected in the heаt capacity curve of HlH in 50 mmol L −1 Tris-HCl buffer, pH 7.2, at a heating rate of 1.0 °C min−1. The calorimetrically observed thermal transition correlates well with the unfolding transition monitored by CD measurements. The two-state kinetic model was used to analyse the process of irreversible thermal denaturation of HlH; Е a of 451 ± 4 kJ mol−1 was calculated. The obtained results on the conformational stability of HlH will facilitate the further investigation of the properties and potential biomedical applications of this respiratory protein.

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of 19F-based CPMG, on-resonance R1ρ and off-resonance R1ρ RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the 19F RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with 19F RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of 19F NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions.Graphic abstract