Conformational Changes Research Articles

Full-length GSDME mediates pyroptosis independent from cleavage.

Gasdermin (GSDM) family proteins, known as the executors of pyroptosis, undergo protease-mediated cleavage before inducing pyroptosis. We here discovered a form of pyroptosis mediated by full-length (FL) GSDME without proteolytic cleavage. Intense ultraviolet-C irradiation-triggered DNA damage activates nuclear PARP1, leading to extensive formation of poly(ADP-ribose) (PAR) polymers. These PAR polymers are released to the cytoplasm, where they activate PARP5 to facilitate GSDME PARylation, resulting in a conformational change in GSDME that relieves autoinhibition. Moreover, ultraviolet-C irradiation promotes cytochrome c-catalysed cardiolipin peroxidation to elevate lipid reactive oxygen species, which is then sensed by PARylated GSDME, leading to oxidative oligomerization and plasma membrane targeting of FL-GSDME for perforation, eventually inducing pyroptosis. Reagents that concurrently stimulate PARylation and oxidation of FL-GSDME, synergistically promoting pyroptotic cell death. Overall, the present findings elucidate an unreported mechanism underlying the cleavage-independent function of GSDME in executing cell death, further enriching the paradigms and understanding of FL-GSDME-mediated pyroptosis.

Single Molecule Thermodynamic Penalties Applied to Enzymes by Whispering Gallery Mode Biosensors.

Optical microcavities, particularly whispering gallery mode (WGM) microcavities enhanced by plasmonic nanorods, are emerging as powerful platforms for single-molecule sensing. However, the impact of optical forces from the plasmonic near field on analyte molecules is inadequately understood. Using a standard optoplasmonic WGM single-molecule sensor to monitor two enzymes, both of which undergo an open-to-closed-to-open conformational transition, the work done on an enzyme by the WGM sensor as atoms of the enzyme movethrough the electric field gradient of the plasmonic hotspot during conformational change has been quantified. As the work done by the sensor on analyte enzymes can be modulated by varying WGM intensity, the WGM microcavity system can be used to apply free energy penalties to regulate enzyme activity at the single-molecule level. The findings advance the understanding of optical forces in WGM single-molecule sensing, potentially leading to the capability to precisely manipulate enzyme activity at the single-molecule level through tailored optical modulation.

Visualizing Single-Molecule Protein Conformational Transitions and Free Energy Landscape.

Monitoring the conformational dynamics of individual proteins is essential to understand the relationship between structure and function in molecular regulatory mechanisms. However, the fast dynamics of single proteins remain poorly understood. Here, we construct a single-molecule sensing platform by introducing plasmonic imaging of single nanoparticles to sense and report the protein conformational changes at the single-molecule level. Tracking the fluctuations of individual nanoparticles with high resolution, we detect and characterize distinct conformational states of molecular chaperone heat shock protein 90 (Hsp90). We also explore the conformational changes of Hsp90 in situ under different nucleotide conditions. Analysis of the conformational fluctuations between the open and closed states of single Hsp90 provides important information on free energy profiles, effective spring constants, and multiphase behaviors. This method offers a strategy to visualize the conformational changes of single proteins in real-time and provides insights into the underlying molecular mechanisms.

Molecular dynamics simulations reveal differences in the conformational stability of FtsZs derived from Staphylococcus aureus and Bacillus subtilis

FtsZ is highly conserved among bacteria and plays an essential role in bacterial cell division. The tense conformation of FtsZ bound to GTP assembles into a straight filament via head-to-tail associations, and then the upper subunit of FtsZ hydrolyzes GTP bound to the lower FtsZ subunit. The subunit with GDP bound disassembles accompanied by a conformational change in the subunit from the tense to relaxed conformation. Although crystal structures of FtsZ derived from several bacterial species have been determined, the conformational change from the relaxed to tense conformation has only been observed in Staphylococcus aureus FtsZ (SaFtsZ). Recent cryo-electron microscopy analyses revealed the three-dimensional reconstruction of the protofilament, in which tense molecules assemble via head-to-tail associations. However, the lower resolution of the protofilament suggested that the flexibility of the FtsZ protomers between the relaxed and tense conformations caused them to form in less-strict alignments. Furthermore, this flexibility may also prevent FtsZs other than SaFtsZ from crystalizing in the tense conformation, suggesting that the flexibility of bacterial FtsZs differs. In this study, molecular dynamics simulations were performed using SaFtsZ and Bacillus subtilis FtsZ in several situations, which suggested that different features of the FtsZs affect their conformational stability.

Conformation of factor Xa in solution revealed by single molecule spectroscopy

All current X-ray structures of fXa are devoid of the γ-carboxyglutamate (Gla) domain and fail to reveal the overall conformation of the free protein. The recent cryo-EM structure of fXa in the prothrombinase complex is the only structure of full length fXa and shows that the Gla domain is positioned at an angle relative to the EGF1 domain. Establish if the curved conformation of fXa revealed by cryo-EM is also present in solution. The conformation of fXa in solution is studied by single molecule Förster resonance energy transfer (smFRET). The conformation of full length fXa in solution is resolved for the first time. The conformation is curved and extremely sensitive to Ca2+. It does not differ significantly from its zymogen form or from that present in the prothrombinase complex free or bound to the physiological substrates prothrombin and meizothrombin. Measurements by smFRET reveal that fXa has a curved conformation in solution, free or bound to physiological ligands, and validate the recent cryo-EM structures of prothrombinase. The drastic conformational changes observed in the absence of Ca2+ suggest that the structural architecture of fXa changes upon administration of vitamin-K antagonists that perturb the interaction of the Gla domain with divalent cations.

Architecture of RabL2-associated complexes at the ciliary base: A structural modeling perspective: Deciphering the structural organization of ciliary RabL2 complexes.

Cilia are slender, micrometer-long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so-called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N-terminal domain of CEP43 forms a homo-dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N-terminal domain of CEP43 from the C-terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2-CEP19 associates with the IFT-B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT-B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled-coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia.

Human Cardiac β-Myosin Powerstroke Energetics: Thin Filament, Pi Displacement and Mutation Effects

The powerstroke of human cardiac β-myosin is an important stage of the cross-bridge cycle that generates force for muscle contraction. However, the starting structure of this process has never been resolved, and the relative timing of the powerstroke and inorganic phosphate (Pi) release is still controversial. In this study, we generated an atomistic model of myosin on the thin filament and utilized metadynamics simulations to predict the absent starting structure of the powerstroke. We demonstrated that the displacement of Pi from the active site during the powerstroke is likely necessary, reducing the energy barrier of the conformation change. The effects of the presence of the thin filament, the hypertrophic cardiomyopathy (HCM) mutation R712L, and the binding of Mavacamten on the powerstroke process were also investigated.

Cryo-EM Structure and Biochemical Analysis of the Human Chemokine Receptor CCR8.

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein-coupled receptor that has emerged as a promising therapeutic target in cancer and autoimmune diseases. In the present study, we solved the cryo-electron microscopy (cryo-EM) structure of the human CCR8-Gi complex in the absence of a ligand at 2.58 Å. Structural analysis and comparison revealed that our apo CCR8 structure undergoes some conformational changes and is similar to that in the CCL1-CCR8 complex structure, indicating an active state. In addition, the key residues of CCR8 involved in the recognition of LMD-009, a potent nonpeptide agonist, were investigated by mutating CCR8 and testing the calcium flux induced by LMD-009-CCR8 interaction. Three mutants of CCR8, Y1133.32A, Y1724.64A, and E2867.39A, showed a dramatically decreased ability in mediating calcium mobilization, indicating their key interaction with LMD-009 and key roles in activation. These structural and biochemical analyses enrich molecular insights into the agonism and activation of CCR8 and will facilitate CCR8-targeted therapy.

Conformation-Driven Responsive 1D and 2D Lanthanide-Metal-Organic Framework Heterostructures for High-Security Photonic Barcodes.

Development of luminescent segmented heterostructures featuring multiple spatial-responsive blocks is important to achieve miniaturized photonic barcodes toward anti-counterfeit applications. Unfortunately, dynamic manipulation of the spatial color at micro/nanoscale still remains a formidable challenge. Here, a straightforward strategy is proposed to construct spatially varied heterostructures through amplifying the conformation-driven response in flexible lanthanide-metal-organic frameworks (Ln-MOFs), where the thermally induced minor conformational changes in organic donors dramatically modulate the photoluminescence of Ln acceptors. Notably, compositionally and structurally distinct heterostructures (1D and 2D) are further constructed through epitaxial growth of multiple responsive MOF blocks benefiting from the isomorphous Ln-MOF structures. The thermally controlled emissive colors with distinguishable spectra carry the fingerprint information of a specific heterostructure, thus allowing for the effective construction of smart photonic barcodes with spatially responsive characteristics. The results will deepen the understanding of the conformation-driven responsive mechanism and also provide guidance to fabricate complex stimuli-responsive hierarchical microstructures for advanced optical recording and high-security labels.

Conformation-Driven Responsive 1D and 2D Lanthanide-Metal-Organic Framework Heterostructures for High-Security Photonic Barcodes.

Development of luminescent segmented heterostructures featuring multiple spatial-responsive blocks is important to achieve miniaturized photonic barcodes toward anti-counterfeit applications. Unfortunately, dynamic manipulation of the spatial color at micro/nanoscale still remains a formidable challenge. Here, a straightforward strategy is proposed to construct spatially varied heterostructures through amplifying the conformation-driven response in flexible lanthanide-metal-organic frameworks (Ln-MOFs), where the thermally induced minor conformational changes in organic donors dramatically modulate the photoluminescence of Ln acceptors. Notably, compositionally and structurally distinct heterostructures (1D and 2D) are further constructed through epitaxial growth of multiple responsive MOF blocks benefiting from the isomorphous Ln-MOF structures. The thermally controlled emissive colors with distinguishable spectra carry the fingerprint information of a specific heterostructure, thus allowing for the effective construction of smart photonic barcodes with spatially responsive characteristics. The results will deepen the understanding of the conformation-driven responsive mechanism and also provide guidance to fabricate complex stimuli-responsive hierarchical microstructures for advanced optical recording and high-security labels.

The Effect of High Intensity Ultrasound on the Quality and Shelf Life of Tilapia (Oreochromis niloticus) Muscle

It has been documented that the shelf life of fishery products is extremely reduced due to microbial development and its endogenous biochemistry. For this reason, food technologists around the world are researching how to reduce the main processes that lead to spoilage. Recently, high-intensity ultrasound (HIU) has had different applications in the food industry because the cavitation effect can inhibit or reduce microbial development as well as cause conformational changes in muscle enzymes. Therefore, in this study, HIU was applied for 30, 60, and 90 min to the tilapia (Oreochromis niloticus) fillet, and subsequently, it was stored on ice for 20 days. During this period, samples were taken every 5 days (day 0, 5, 10, 15, and 20), and moisture content, pH, total volatile base (TVB-N), non-protein nitrogen (NPN), texture, electrophoresis, color, and microbiological analyses (mesophiles and psychrophiles) were determined. No significant changes (p ≥ 0.05) were observed in the moisture content, pH, and the L* parameter, while a significant decrease (p < 0.05) in TVB-N (from 29.67 to 15.09), NPN (from 0.39 to 0.27%), and texture (from 4.88 to 2.69 N) were found. On the other hand, an increase (p < 0.05) in a* (from 2.02 to 4.27) and b* (from 10.66 to 12.45) parameters, as well as total mesophile count (from 2.48 to 6.52 log CFU/g) were detected due to the application of ultrasound. The results suggest that the application of this treatment represents a viable alternative to increase the shelf life and quality of tilapia fillets stored on ice.

Structural and Electronic Evolution of Ethanolamine upon Microhydration: Insights from Hyperfine Resolved Rotational Spectroscopy.

Ethanolamine hydrates containing from one to seven water molecules were identified via rotational spectroscopy with the aid of accurate quantum chemical methods considering anharmonic vibrational corrections. Ethanolamine undergoes significant conformational changes upon hydration to form energetically favorable hydrogen bond networks. The final structures strongly resemble the pure (H2O)3-9 complexes reported before when replacing two water molecules by ethanolamine. The 14N nuclear quadrupole coupling constants of all the ethanolamine hydrates have been determined and show a remarkable correlation with the strength of hydrogen bonds involving the amino group. After addition of the seventh water molecule, both hydrogen atoms of the amino group actively contribute to hydrogen bond formation, reinforcing the network and introducing approximately 21-27% ionicity towards the formation of protonated amine. The findings highlight the critical role of microhydration in altering the electronic environment of ethanolamine, enhancing our understanding of amine hydration dynamics.

Structural and Electronic Evolution of Ethanolamine upon Microhydration: Insights from Hyperfine Resolved Rotational Spectroscopy.

Ethanolamine hydrates containing from one to seven water molecules were identified via rotational spectroscopy with the aid of accurate quantum chemical methods considering anharmonic vibrational corrections. Ethanolamine undergoes significant conformational changes upon hydration to form energetically favorable hydrogen bond networks. The final structures strongly resemble the pure (H2O)3-9 complexes reported before when replacing two water molecules by ethanolamine. The 14N nuclear quadrupole coupling constants of all the ethanolamine hydrates have been determined and show a remarkable correlation with the strength of hydrogen bonds involving the amino group. After addition of the seventh water molecule, both hydrogen atoms of the amino group actively contribute to hydrogen bond formation, reinforcing the network and introducing approximately 21-27% ionicity towards the formation of protonated amine. The findings highlight the critical role of microhydration in altering the electronic environment of ethanolamine, enhancing our understanding of amine hydration dynamics.

Revealing the deterioration mechanism in gelling properties of pork myofibrillar protein gel induced by high-temperature treatments: Perspective on the protein aggregation and conformation

The purpose of the present study was to investigate the mechanism of gel deterioration of myofibrillar proteins (MP) gels induced by high-temperature treatments based on the protein aggregation and conformation. The results showed that the gel strength and water holding capacity of MP obviously increased and then decreased as the temperature increased, reaching the maximum value at 80 °C (P < 0.05). The microstructure analysis revealed that appropriate temperature (80 °C) contributed to the formation of a more homogeneous, denser, and smoother three-dimensional mesh structure when compared other treatment temperatures, whereas excessive temperature (95 °C) resulted in the formation of heterogeneous and large protein aggregates of MP, decreasing the continuity of gel networks. This was verified by the rheological properties of MP gels. The particle size (D4,3 and D3,2) of MP obviously increased with larger clusters at excessive temperature, and the surface hydrophobicity of MP decreased (P < 0.05), which has been linked to the formation of soluble or insoluble protein aggregates. Tertiary structure and secondary structure results revealed that the proteins had a tendency to be more stretched under higher temperature treatments, which resulted in a decrease in covalent interactions and non-covalent interactions, fostering the over-aggregation of MP. Therefore, our present study indicated that the degradation of MP gels treated at high temperatures was explained by protein aggregation and conformational changes in MP.

Structural and Electronic Evolution of Ethanolamine upon Microhydration: Insights from Hyperfine Resolved Rotational Spectroscopy

Ethanolamine hydrates containing from one to seven water molecules were identified via rotational spectroscopy with the aid of accurate quantum chemical methods considering anharmonic vibrational corrections. Ethanolamine undergoes significant conformational changes upon hydration to form energetically favorable hydrogen bond networks. The final structures strongly resemble the pure (H2O)3‐9 complexes reported before when replacing two water molecules by ethanolamine. The 14N nuclear quadrupole coupling constants of all the ethanolamine hydrates have been determined and show a remarkable correlation with the strength of hydrogen bonds involving the amino group. After addition of the seventh water molecule, both hydrogen atoms of the amino group actively contribute to hydrogen bond formation, reinforcing the network and introducing approximately 21‐27% ionicity towards the formation of protonated amine. The findings highlight the critical role of microhydration in altering the electronic environment of ethanolamine, enhancing our understanding of amine hydration dynamics.

The crystal structure of Shethna protein II (FeSII) from Azotobacter vinelandii suggests a domain swap.

The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239-247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.

Chemoenzymatic Synthesis of Fluorinated Mycocyclosin Enabled by the Engineered Cytochrome P450-Catalyzed Biaryl Coupling Reaction.

Installing fluorine atoms onto natural products holds great promise for the generation of fluorinated molecules with improved or novel pharmacological properties. The enzymatic oxidative carbon-carbon coupling reaction represents a straightforward strategy for synthesizing biaryl architectures, but the exploration of this method for producing fluorine-substituted derivatives of natural products remains elusive. Here, in this study, we report the protein engineering of cytochrome P450 from Mycobacterium tuberculosis (MtCYP121) for the construction of a series of new-to-nature fluorine-substituted Mycocyclosin derivatives. This protocol takes advantage of a "hybrid" chemoenzymatic procedure consisting of tyrosine phenol lyase-catalyzed fluorotyrosine preparation from commercially available fluorophenols, intermolecular chemical condensation to give cyclodityrosines, and an engineered MtCYP121-catalyzed intramolecular biphenol coupling reaction to complete the strained macrocyclic structure. Computational mechanistic studies reveal that MtCYP121 employs Cpd I to abstract a hydrogen atom from the proximal phenolic hydroxyl group of the substrate to trigger the reaction. Then, conformational change makes the two phenolic hydroxyl groups close enough to undergo intramolecular hydrogen atom transfer with the assistance of a pocket water molecule. The final diradical coupling process completes the intramolecular C-C bond formation. The efficiency of the biaryl coupling reaction was found to be influenced by various fluorine substitutions, primarily due to the presence of distinct binding conformations.

Studies of Light Scattering by Hemoglobin Molecules under the Effect of Iron (III) Chloride and Various pH Levels

A lot of literature sources discuss diseases associated with the hemoglobin protein [1, 2]. In the modern world, more than 800 million people suffer from anemia [3]. Assessing the level of hemoglobin in the circulatory system is one of the ways to diagnose anemia. Hemoglobin protein is also a promising source of bioactive peptides [4]. The aim of the work was to identify the reasons leading to conformation changes of this protein. It still remains unclear what exactly causes disruption of its functionality. Human hemoglobin from Sigma H7379 was studied in the work; all experiments were conducted on a dynamic light scattering spectrometer — Photocor Complex. Experimental data were obtained using optical methods of static and dynamic light scattering. The work included an analysis of the behaviour of hemoglobin protein molecules in aqueous and aqueous-saline solutions with changes in solution parameters (pH, addition of iron chloride III). At values of pH&lt; (3.56±0.15) and pH&gt; (10.4±0.2), the hemoglobin molecule underwent conformational changes, resulting in the disintegration of the quaternary structure into αβ−dimers and individual α− and β−globules. As a result of the study, it was found that the addition of FeCl3 to aqueous solutions of hemoglobin increases the size and mass of scattering particles, which can be explained by the adsorption of Fe3+ ions on the protein surface. However, upon reaching a certain concentration of FeCl3, the pH of the solution was lowered to such an extent that it caused conformational changes in hemoglobin, leading to the disintegration of its quaternary structure. These results can be taken into account when creating medicinal drugs for the treatment of anemia and other diseases associated with the hemoglobin protein.

In silico Investigation on the Structural Insights into the Binding of Squalamine Inhibitor with Membrane-Bound Α-Synuclein

Background: Parkinson's disease (PD) and its associated symptoms are closely associated with the self-assembly of α-Synuclein (α-Syn). Squalamine is a naturally occurring chemical substance with established antiviral and anticancer properties, and its profound impact on the α- Syn aggregation both in vivo and in vitro is well studied. Examining its interaction with lipid vesicles, which are known to encourage nucleation, can signify the mechanism of action of squalamine. The squalamine molecule is believed to displace α-Syn from the surfaces of the lipid vesicles, therefore preventing the initial steps in the process of aggregation. Additionally, the squalamine molecule reduces the harmful effects of α-Syn oligomers in human neuroblastoma cells by preventing them from interacting with lipid membranes. Objective: The aim of this study was to perform computational investigation of the conformational changes of membrane-bound α-Syn in the presence of squalamine inhibitor molecule objective: Computational investigation of the conformational changes of membrane-bound α-Synuclein in the presence of squalamine inhibitor molecule Method: Molecular Dynamics (MD) trajectory analysis was carried out to study the structural change of the α-Syn-squalamine conformers as a function of simulation time. The percentage of the secondary structural components of the α-Syn-squalamine complex was determined. Optimization of small molecule inhibitors was carried out using Density Functional Theory (DFT) analysis. Additionally, the values of electrophilicity (ω), nucleophilicity (N), Electron affinity (EA), and ionization potential (IP) were calculated. Results: The docking of the α-Syn-squalamine complex revealed the binding site and the best structure was selected based on the highest docking vina score (-5.8), and the contact residues were listed. From the conformational snapshots of the α-Syn-squalamine complex, it was evident that the α-Syn remained stable, maintaining its integrity throughout the simulation. The α-helical content was found to be retained from the secondary structural content analysis. The ω and N of the squalamine molecule were calculated to be -0.84 and 3.25, respectively. Conclusion: Our findings suggest that in the presence of a squalamine inhibitor molecule, α-Syn adopts a helical conformation that ensures stability and may indicate that the squalamine molecule causes gradual displacement of α-Syn across different regions within the lipid membrane.

Towards interpretable Cryo-EM: disentangling latent spaces of molecular conformations

Molecules are essential building blocks of life and their different conformations (i.e., shapes) crucially determine the functional role that they play in living organisms. Cryogenic Electron Microscopy (cryo-EM) allows for acquisition of large image datasets of individual molecules. Recent advances in computational cryo-EM have made it possible to learn latent variable models of conformation landscapes. However, interpreting these latent spaces remains a challenge as their individual dimensions are often arbitrary. The key message of our work is that this interpretation challenge can be viewed as an Independent Component Analysis (ICA) problem where we seek models that have the property of identifiability. That means, they have an essentially unique solution, representing a conformational latent space that separates the different degrees of freedom a molecule is equipped with in nature. Thus, we aim to advance the computational field of cryo-EM beyond visualizations as we connect it with the theoretical framework of (nonlinear) ICA and discuss the need for identifiable models, improved metrics, and benchmarks. Moving forward, we propose future directions for enhancing the disentanglement of latent spaces in cryo-EM, refining evaluation metrics and exploring techniques that leverage physics-based decoders of biomolecular systems. Moreover, we discuss how future technological developments in time-resolved single particle imaging may enable the application of nonlinear ICA models that can discover the true conformation changes of molecules in nature. The pursuit of interpretable conformational latent spaces will empower researchers to unravel complex biological processes and facilitate targeted interventions. This has significant implications for drug discovery and structural biology more broadly. More generally, latent variable models are deployed widely across many scientific disciplines. Thus, the argument we present in this work has much broader applications in AI for science if we want to move from impressive nonlinear neural network models to mathematically grounded methods that can help us learn something new about nature.