Moreover, facile preparation of metallosalen-covalent organic frameworks with good crystallinity remains considerably challenging. Herein, we report a series of metallosalen-covalent organic frameworks produced via a one-step synthesis strategy that does not require vacuum evacuation. Metallosalen-covalent organic frameworks possessing controllable coordination environments of mononuclear and binuclear metal sites are obtained and act as photocatalysts for tunable syngas production from CO2. Metallosalen-covalent organic frameworks obtained via one-step synthesis exhibit higher crystallinity and catalytic activities than those obtained from two-step synthesis. The optimal framework material containing cobalt and triazine achieves a syngas production rate of 19.7 mmol g−1 h−1 (11:8 H2/CO), outperforming previously reported porous crystalline materials. This study provides a facile strategy for producing metallosalen-covalent organic frameworks of high quality and can accelerate their exploration in various applications.Metallosalen-covalent organic frameworks have recently gained attention in photocatalysis. However, their use in CO Here, the authors report a gram-scale, one-step synthesis of metallosalen-covalent organic frameworks -COFs) without vacuum evacuation. M-COFs exhibit good crystallinity and adjustable activities in CO2 photoreduction to syngas. Metallo-covalent organic frameworks (M-COFs) represent a fusion of organometallic and polymer chemistry that might be instrumental in creating excellent heterogeneous single-metal-site catalysts 3. In M-COFs, active metal sites are periodically integrated into extended frameworks that can effectively protect metal sites from aggregation9. Furthermore, well-defined coordination environments of metals can be conveniently achieved in M-COFs11. Determining the precise position and microenvironments of metal species in M-COFs is useful for understanding the relationships between structures and properties in catalysis. Subsequently, the catalytic performance of M-COFs can be optimised by regulating the metal species and their coordination environments15. Salen, porphyrins, and 2, 2’-bipyridine (bipy) are three chelating ligands commonly used to coordinate metal ions that form metal complexes. Among them, the salen moiety is one of the most essential ligands in coordination chemistry because of its ability to form various metal complexes ) that can be utilised as the most powerful homogeneous catalysts for various organic transformations15. The metallosalen-covalent organic frameworks -COFs), which embed M, retain both the unique catalytic activities of M and the extended porous frameworks of COFs, endowing them with promising capabilities of heterogeneous SMS catalysts18. However, using M-COFs in photocatalytic CO2 reduction has not been reported.Covalent organic frameworks (COFs) are a burgeoning class of porous organic polymers that have garnered considerable attention in various fields, such as catalysis, gas adsorption and separation, energy storage and sensing19. The metalation procedure inevitably results in increased time consumption, lower yields and decreased COF crystallinity due to the framework’s collapse20. Moreover, producing highly crystalline M-COF requires high-purity multidentate organic ligands, which increases the difficulty of synthesis. In most cases, the ampoule containing ligands, solvents and catalysts must be flash-frozen in a liquid nitrogen bath, evacuated, and flame-sealed21. In general, only tens of milligrams of highly crystalline M-COFs can be obtained. Harsh reaction conditions and low yields severely limit large-scale production and exploration for various applications. Thus, developing a facile synthetic method for M-COFs with high yield is vital and an attractive strategy is a multicomponent one-step reaction with more reversible chemistry24.Furthermore, the synthesis of M-COFs usually involves at least a two-step process that requires post-synthetic metalation2O2 (e.g. Zn-TAPB-COF-1) and binuclear metal site M-COFs with metal coordination environments of M-N2O2-M-N2O2 (e.g. ZnZn-TAPB-COF-1). We also allowed Co instead of Zn to be embedded in the frameworks. The precise position and coordination environments for the metal sites were determined via X-ray absorption fine structure (XAFS) of synchrotron radiation analysis. Finally, the synthesised M-COFs were applied as photocatalysts in CO2 reduction. Changing the metal species, coordination environments, and ligands in M-COFs could easily achieve tunable syngas proportions. In particular, the M-COFs obtained via the one-step strategy exhibited considerably stronger crystallinity and higher photocatalytic activities than those obtained via two-step synthesis.Herein, we report the preparation of M-COFs via a one-step synthesis in autoclaves without vacuum deaeration, thus yielding gram-scale M-COFs. We fabricated mononuclear metal site M-COFs with metal coordination environments of M-No-DCB), N, N-dimethylformamide (DMF) and an acetic acid (3 M HOAc) mixture were added as reaction solvents and catalysts. The autoclave was further heated under solvothermal conditions . For the two-step synthesis of M-COFs-2, M-CHO or MM-CHO (M = Zn and Co) were first synthesised benzene (TAPB) or 2, 4, 6-tris(4-aminophenyl)−1, 3, 5-triazine (TAPT), were mixed and heated in the solvent system HOAc/o-DCB/n-BuOH at 120 °C for 3 days. M-COFs were obtained after washing with ethanol and tetrahydrofuran (THF). Further, the wet M-COFs were soaked in methanol and liquid CO2  for 24 h. Finally, the M-COFs were dried with supercritical CO2  for 24 h. The M-COFs yields obtained via the one-step route were higher than those obtained via the two-step route -COFs from the simple alicylaldehyde ligand, i.e. one-step and two-step syntheses Fig. . The onesed Fig. –S4. Subssed Fig. , includite Table . Zn-TAPBte Table  and binute Table . Apart fte Table .Fig. 1Sc25. Given that only 1D fringes were observed and the honeycomb structures were absent in their TEM images, AA serrated models without 6-fold or 3-fold symmetry were more reasonable modes for the synthesised M-COFs. Furthermore, the unit-cell parameters of M-COFs-1 were optimised by a universal force field (Zn-TAPB-COF-1: a = 51.444(1) Å, b = 51.235(8) Å, c = 7.373(2) Å, α = 74.6458°, β = 98.9531°, γ = 121.1356°; ZnZn-TAPB-COF-1: a = 51.291(3) Å, b = 51.194(2) Å c = 7.137(7) Å, α = 88.8966°, β = 92.1975°, γ = 119.7291°; Co-TAPT-COF-1: a = 50.573(2) Å, b = 50.127(7) Å, c = 7.087(5) Å, α = 74.4833°, β = 97.2283°, γ = 120.9587°)20. In addition, fractional atomic coordinates for the unit cells of Zn-TAPB-COF-1, ZnZn-TAPB-COF-1 and Co-TAPT-COF-1 are summarised in Tables 26. The stronger crystallinity of M-COFs from the one-step synthesis could be attributed to multicomponent co-existence, which provided a larger space for the reversible formation of imine bonds and structural self-healing of M-COFs27. Moreover, the possibility of scaling up this one-step synthesis for crystalline M-COFs was investigated. Crystalline Co-TAPB-COF-1 powder of 2.61 g with a single synthesis was also prepared from the one-step synthesis -COFs were characterised using powder X-ray diffraction (PXRD) technology. All M-COFs obtained from the one-step synthesis showed sharp diffraction peaks Fig. . Simulatsis Fig.   NMR spectra with characteristic carbon signals at 158–163 ppm further confirmed the formation of –C = N– in M-COFs -COFs obtained from different synthesis methods possess the same chemical structures. Thermogravimetric analysis curves showed that all M-COFs could maintain thermal stability up to 300 °C -COFs  of these M-COFs were distributed from 320 to 754 m2 g−1 (Table 20. Micropores (1.1 nm) and mesopores (3.8 nm) co-existed in M-COF-2 synthesised by the two-step synthesis28. The differences in pore sizes between M-COFs from one-step and two-step synthesis might be caused by partial pore collapse and lower crystallinity in M-COFs-229. The theoretical pore size distributions of Zn-TAPB-COF-1 (~3.7 nm) and ZnZn-TAPB-COF-1 (~3.8 nm) were calculated using the Zeo + + programme -COFs were then verified using Fourier transform infrared (FT-IR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. The FT-IR spectra showed typical bands for imine bonds (–C = N–) at 1608–1639 cm−1 Figs. –S12. SubFs Figs. –S16. The °C Fig. . N2 adsoFs Figs. –S23. For−1 Table . Non-locme Figs. , 630. Th32. The Zn contents in Zn-TAPB-COF-1 and ZnZn-TAPB-COF-1, as detected via inductively coupled plasma atomic emission spectroscopy (ICP), were 8.23 wt% and 8.31 wt%, respectively. Co contents in Co-TAPB-COF-1 and Co-TAPT-COF-1 were 9.47 wt% and 10.34 wt%, respectively. Such high metal loading with high dispersion was rare because aggregation to clusters or nanoparticles always occurred easily at metal contents above 2 wt%33. Abundant and highly dispersed metal species in these M-COFs were expected to exhibit excellent catalytic activities. Further, XANES and X-ray photoelectron spectroscopy (XPS) were used to confirm the electronic state of the metals in M-COFs  further visualised the metal species’ coordination forms in M-COFs  for Zn-TAPB-COF-1 could be assigned to the Zn–O or Zn–N bonds. As for ZnZn-COF-TAPB-1, there were two apparent shells in 4.5 Å−1 (x-axis: 1.5 Å) and 9 Å−1 (x-axis: 4.5 Å), which could be assigned to Zn–O (N) and Zn–O–Zn bonds. For Co-TAPT-COF-1, the WT maximum at 6.36 Å−1 (x-axis: 1.43 Å) could be assigned to the Co−O or Co-N bonds -COFs, aberration-corrected transmission electron microscopy (AC-TEM), X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure of synchrotron radiation (EXAFS) were conducted. AC-TEM images of Zn-TAPB-COF-1, ZnZn-TAPB-COF-1, and Co-TAPT-COF-1 all exhibited high-density bright dots without agglomerates, indicating their uniform isolated metal distribution Fig. 31,32. ThFs Figs. , 25. In Fs Figs. 34. The CCoO Fig. , indicatCoO Fig. . SubsequCoO Fig. . The FT-CoO Fig. 31,33. No-1 Table . FT-EXAFnds Fig. . As for six Fig. , suggestOFs Fig. . The WT nds Fig. . These c37. The influence of synthetic routes on M-COF morphology has rarely been studied. Considering that the synthesis strategies mentioned above have considerable impact on the crystallinity of M-COFs, we hypothesise that these strategies might also affect the morphologies of M-COFs. As shown in Figs. 2 absorption ability -COFs resulted from differing reaction environments of the two syntheses strategies, which could profoundly affect the formation of imine bonds, crystal nucleation, and growth38. Fragments in the one-step synthesis tended to self-assemble into M-COFs with larger scales because of the co-existence of all raw materials. In the stepwise synthesis, 4-hydroxyisophthalaldehyde  and ethylenediamine were not present in the second step. Thus, condensation reactions in the second step occurred only on the highly dispersed nuclei generated from the first step, limiting the larger-scale growth of M-COFs35. In addition to the synthesis strategies, coordination environments, ligands and metal species affected the morphology of M-COF superstructures.Thus far, precise construction of M-COFs superstructures without templates remains unclearity Fig. . Accordi2 reduction to syngas (the mixture of CO and H2 that can be converted into a variety of fuels) has become a hot research trend owing to its mild reaction conditions, high safety level and environmental friendliness40. However, the synthesis of syngas with controlled proportions for different downstream products remains a great challenge, hindering this method’ implementation41. Notably, the structure–performance relationships of photocatalysts remain unrevealed. Here, we demonstrated highly tunable production of syngas with synthesised M-COFs as photocatalysts  and H2 rate of 11.31 mmol g−1 h−1 (113.10 μmol h−1) in 4 h  centres in M-COFs were expected to act as excellent CO2 adsorption and activation sites44. Notably, Co-TAPT-COF-2 exhibited a CO production rate of 3.50 mmol g−1 h−1 and a H2 production rate of 4.72 mmol g−1 h−1, revealing that the synthesis routes of M-COFs considerably affected their photocatalytic activity  than Co-TAPT-COF-1  was employed as the source gas  signal when using 13CO2 as the source gas. No 12CO (m/z = 28) was detected, demonstrating that all the CO originated from CO247. To assess the stability of Co-TAPT-COF-1, recycling experiments and post-catalysis characterisations, including PXRD, FT-IR and N2 adsorption–desorption isotherms, were further conducted -TAPT , were calculated by DFT calculations -TAPT was the rate-determining step (RDS) for CO production -COFs were conducted to investigate their photoelectric properties. The UV–vis diffuse reflectance spectra revealed that all Co-COFs exhibited broad visible light absorption  positions -COFs in photocatalysis, density functional theory (DFT) calculations and spectroscopic characterisations were performed. First, free energies for the conversion of COons Fig. . Accordiion Fig. . More paion Fig. . When coion Fig. . The banion Fig. . Mott–Scion Fig. . The posons Fig. . In addions Fig. , indicat2O2 and M–N2O2–M–N2O2) were fabricated and the structures were confirmed by XAFS spectra. The M-COFs obtained from this one-step strategy showed considerably higher crystallinity and larger-scale superstructures than those obtained from the two-step synthesis, exhibiting superior photocatalytic performance. In particular, Co-TAPT-COF-1 exhibited distinct nanotube superstructures and showed the highest activity towards syngas production from CO2 photoreduction . The ratio of n(H2)/n(CO) was widely tunable by regulating the M-COF structures and synthesis routes. These results provided insights into the structure–property relationships and design principles of high-efficiency photocatalysts. Moreover, the high yield and mild conditions of this one-step synthesis laid the foundation for exploring the industrial applications of M-COFs.In conclusion, the one-step synthesis strategy proposed in this work provided a facile and scalable method for preparing M-COFs . M-COFs with different metal coordination environments  were added to a 50-mL two-necked flask. Further, degassed ethanol (15 mL) and ethylenediamine  were added to the flask. The mixture was stirred at 50 °C under N2 atmosphere for 5 h. Upon cooling, Zn-CHO was obtained by washing with cold methanol three times (yield: 90%). Co-CHO was synthesised similarly to Zn-CHO except that Zn(OAc)2·2H2O was replaced with Co(OAc)2·4H2O (yield: 84%).4-hydroxyisophthalaldehyde  and Zn(OAc)2·2H2O  were added to a 50-mL two-necked flask. Further, degassed ethanol (20 mL) and ethylenediamine  were added to the flask. The mixture was stirred at 50 °C under a N2 atmosphere for 5 h. Upon cooling, ZnZn-CHO was obtained by washing with cold methanol three times (yield: 53%).2-hydroxybenzene-1, 3, 5-tricarbaldehyde  and Zn(OAc)2·2H2O  were added into a 20-mL Teflon-sealed autoclave. Further, 4 mL of ethanol and ethylenediamine  were added. After sonication for 5 min, 2 mL of o-DCB, 0.5 mL of DMF, and 1.6 mL of HOAc (3 M) were added to the above suspension. After another 5 min sonication, 1, 3, 5-tri(4-aminophenyl) benzene  was added to the reaction system. The resulting mixture was sonicated for 15 min and heated at 120 °C for 72 h. Finally, yellow powder was obtained after washing with ethanol and THF three times. The yellow powder was soaked in methanol and then in liquid CO2  for 24 h. Finally, the powder was dried using supercritical CO2  treatment for 24 h (yield: 87%). Additionally, Zn-TAPT-COF-1 was synthesised similarly to Zn-TAPB-COF-1, except that TAPB was replaced with TAPT (yield: 67%). Co-TAPB-COF-1 was synthesised similarly to Zn-TAPB-COF-1, except that Zn(OAc)2·2H2O was replaced with Co(OAc)2·4H2O (yield: 83%).In a one-step synthesis of Zn-TAPB-COF-1, 4-hydroxyisophthalaldehyde  and Zn(OAc)n-BuOH, 1.6 mL of o-DCB, and 0.3 mL of HOAc (3 M) were added to the reaction system. The autoclave was then heated in an oven at 120 °C for 72 h, and yellow Zn-TAPB-COF-2 powder was obtained after being washed with ethanol and THF three times. The powder was soaked in methanol and then in liquid CO2  for 24 h. Finally, the powder was dried using scCO2  treatment for 24 h (yield: 67%). Zn-TAPT-COF-2 was synthesised similarly to Zn-TAPB-COF-2, except that TAPB was replaced with TAPT (yield: 66%). Similarly, Co-TAPB-COF-2 was synthesised in a similar way with Zn-TAPB-COF-2, except that Zn-CHO was replaced by Co-CHO (yield: 53%).In a two-step synthesis of Zn-TAPB-COF-2, Zn-CHO was first synthesised. Further, Zn-CHO  and TAPB  were added to a 20-mL Teflon-sealed autoclave. Subsequently, 6.4 mL of 2·2H2O  were added to a 20-mL Teflon-sealed autoclave. Further, 4 mL of ethanol and ethylenediamine  were added. Following sonication for 5 min, 2 mL of o-DCB and 0.5 mL of DMF and 1.6 mL of HOAc (3 M) were added to the suspension. After sonication for another 10 min, 1, 3, 5-tri(4-aminophenyl) benzene  was added to the reaction system. The obtained suspension was sonicated for 15 min and then heated at 120 °C for 72 h. Yellow–brown powder was obtained after washing with ethanol and THF three times. The powder was soaked in methanol and then in liquid CO2  for 24 h. Finally, scCO2  treatment for 24 h was used to dry the powder (yield: 81%). In addition, ZnZn-TAPT-COF-1 was synthesised similarly to ZnZn-TAPB-COF-1, except that TAPB was replaced with TAPT (yield: 75%).For one-step synthesis of ZnZn-TAPB-COF-1 sample, 2-hydroxybenzene-1, 3, 5-tricarbaldehyde  and Zn(OAc)n-BuOH, 1.6 mL of o-DCB, and 0.7 mL of HOAc (3 M) were added to the reaction system, which was then heated at 120 °C for 72 h. Yellow-brown ZnZn-TAPB-COF-2 powder was obtained after washing with ethanol and THF three times. The powder was soaked in methanol and then in liquid CO2  for 24 h. Finally, scCO2  treatment for another 24 h was used to dry the powder (yield: 39%). In addition, ZnZn-TAPT-COF-2 was synthesised similarly to ZnZn-TAPB-COF-2, except that TAPB was replaced with TAPT (yield: 36%).For two-step synthesis of ZnZn-TAPB-COF-2, ZnZn-CHO was first synthesised. Further, ZnZn-CHO  and TAPB  were added to a 20-mL Teflon-sealed autoclave. Moreover, 6.4 mL of 13C solid-state spectra were recorded using a Varian Infinity-400 spectrometer. TGA was performed using a Netzsch Model STA 449 C micro-analyser under N2 atmosphere from 25 °C to 900 °C. N2 absorption and desorption isotherms were obtained using a quantachrome automated surface area & pore size analyzer, and the corresponding pore size distributions were estimated through nonlocal density functional theory (NLDFT). XPS spectra were acquired from an ESCALAB 250XI with a monochromatic Al Kα X-ray source. Products in a 13C labelled photocatalytic experiment were detected using a SHIMADZU GC-MS-QP2010 SE equipped with a ShiCap-PLOT Q column.Powder X-ray diffraction was performed on a Rigaku D-MAX 2500/PC. FT-IR spectra were performed on a Bruker VERTEX 70 V. 2 reduction was performed using the Labsolar-6A system  connected using Agilent 8890 gas chromatography. In a typical experiment, M-COFs (10 mg) were dispersed in a H2O/ TEOA/ CH3CN mixture  and then [Ru(bpy)3]Cl2·6H2O (60 mg) was added. Further, the mixture was thoroughly degassed and then backfilled with pure CO2 to a pressure of 75 KPa three times. A 300 W xenon lamp with a UV cut-off filter (λ ≥ 420 nm) was used as the light source. During the photocatalytic process, the mixture was vigorously stirred with a magnetic stirrer (400 rpm) and cooled via circulating water to maintain a temperature of approximately 25 °C. The influence of [Ru(bpy)3]Cl2·6H2O contents was tested with Co-TAPT-COF-1 (10 mg) under the same solvent system as above. Different TEOA and H2O proportions  were also investigated using Co-TAPT-COF-1 (10 mg) and [Ru(bpy)3]Cl2·6H2O (60 mg), respectively. To conduct the recirculation test, Co-TAPT-COF-1 (10 mg) and [Ru(bpy)3]Cl2·6H2O (60 mg) were dispersed in a H2O/TEOA/CH3CN mixture . After photocatalytic testing for 4 h, the sample was filtered through an organic filter membrane and used for subsequent recycling tests. For the AQE test, Co-TAPT-COF-1 (17 mg) and [Ru(bpy)3]Cl2·6H2O (60 mg) were dispersed in a H2O/TEOA/CH3CN mixture . Further, the mixture was thoroughly degassed and then backfilled with pure CO2 to 75 KPa three times. A 50 mW cm−2 xenon lamp with a UV band-pass filter (λ = 420 nm) was used as the light source.Photocatalytic CO50. The van der Waals correction of Grimme’s D3 scheme52 was incorporated to describe noncovalent interactions between the adsorbent and the adsorbate. The 6-31 G (d) basis set was used for the nonmetal atoms , and the SDD effective core potential was used for the metal atoms. For each geometric stationary point, single-point energy calculations were performed using an extended 6-31 + + G basis set for the nonmetal atoms and the SMD solvation model was used to determine the effect of the solvent (water)53. Thermal corrections to the free energy of each reactant, product and intermediate state were calculated via frequency calculations (298.15 K and 1 atm) based on optimised geometry. All DFT calculations were performed using Gaussian 16 software54.In the DFT calculations, three model catalysts, including M-TAPT , were used as calculated models. Geometry optimisations and frequency calculations were conducted using the B3LYP functional2 reduction process, as well as in intermediate states were proposed and evaluated. The reaction scheme is as follows:+ + e−) at standard conditions was assumed to be the energy of 1/2H2. The free energy change is described by the following expressions:The reactant and product states in the photocatalytic COSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Source Data


Then, a novel crystal plasticity model was proposed for describing the grains’ behaviour and reflecting the crystals’ actual deformation mechanism under warm forming conditions. Afterward, to clarify the in-grain deformation and link the mechanical behaviour of AA2060-T8 with its microstructural state, RVE elements were created to represent the microstructure of AA2060-T8, where several finite elements discretized every grain. A remarkable accordance was observed between the predicted results and their experimental counterparts for all testing conditions. This signifies that coupling CH with CP modelling can successfully determine the warm deformation behaviour of AA2060-T8  under different working conditions.This study aimed to propose a new approach for predicting the warm deformation behaviour of AA2060-T8 sheets by coupling computational homogenization (CH) with crystal plasticity (CP) modeling. Firstly, to reveal the warm deformation behaviour of the AA2060-T8 sheet, isothermal warm tensile testing was accomplished using a Gleeble-3800 thermomechanical simulator at the temperatures and strain rates that varied from 373 to 573 K and 0.001 to 0.1 s One of the most critical lightweight metallic materials used recently in aircraft, military, and aerospace industries is the third generation Al-Li alloys due to their remarkable mechanical and physical properties compared with conventional Al alloys. Adding Li caused these outstanding properties, impacting the modulus of elasticity and weight reduction. Adding 1% of Li reduced and enhanced the density and modulus of elasticity of Al alloy by 3% and 6%, respectively ,3. Al-Li−1 are the optimum hot forming conditions (temperatures and strain rates) for AA2060-T8 alloy sheets. After that, Gao et al. [−1 and 470 °C are the optimum HFQ conditions (strain rate and temperature) to manufacture sound AA2060-T8 components.So far, little investigations have been accomplished to figure out the flow behaviour and deformation mechanisms of this alloy at high temperatures. Abd El-Aty et al. ,23 inveso et al.  combinedo et al.  to examiFrom the above discussion, it is clear that figuring out the flow behaviour of AA2060 at warm forming conditions experimentally and theoretically was not yet studied. The flow behaviours of Al alloys under warm forming conditions are complex since they depend on many factors  . These kThree essential features are required to simulate and predict the macroscopic mechanical behaviour by coupling CH and CP modeling. The first feature is the constitutive equation that characterizes the grains’ behaviour for reflecting the crystal deformation mechanisms. The second feature represents the actual microstructure AA2060-T8 sheets using RVE. The third feature is the material parameters that are crucial for characterizing the behaviours of grains correctly. This investigation used CP modeling as the constitutive model for AA2060 sheet grains. In the proposed CP model, the total dislocation density was decomposed into edge and screw components to consider DRV caused by a cross slip of the screw dislocations at low homologous temperatures. The experimental stress-strain curves characterized the material parameter of the proposed CP model. After that, the proposed constitutive model was implemented using a monolithic time-integration algorithm-based backward Euler method. For considering the in-grain deformation behaviours, the homogenization process used the representative volume element (RVE) for representing the real sheet metal microstructure. For constructing RVE, the initial microstructures and micro-textures were measured by electron backscatter diffraction (EBSD). In addition, periodic boundary conditions (PBC) were developed for considering geometrical and deformation-induced anisotropy. The accuracy of the proposed approach (coupling CH with CP modeling) for predicting the warm deformation behaviour of AA2060 at different conditions was verified by comparing the experimental and predicted results. Additional verification was performed by calculating a set of statistical parameters for quantitatively evaluating the reliability and measuring the predictability of the proposed approach.−1 using the sample depicted in The material utilized in the current study was rolled sheets of AA2060-T8. A Gleeble-3800 simulator was utilized to accomplish the isothermal warm tensile tests at 373, 423, 473, 523, as well as 573 K and −1, 10,000 °Cs−1, and 2000 mms−1, respectively. Furthermore, the Gleeble-3800 simulator is outfitted with a control system for imposing the aggressive decay of the speed of the actuator to achieve a constant strain rate. The setup of the Gleeble-3800 simulator used in this study is shown in The Gleeble-3800 simulator was designed to perform the physical simulations of thermomechanical processes at a maximum heating rate, quenching rate, and stroke rate of 10,000 °CsSeveral items are necessary to predict the warm deformation behaviour of AA2060 via coupling CH with CP modelling. These items are a new constitutive equation describing the grain’s behaviour for reflecting the deformation mechanism of crystals under warm working conditions, RVE for representing grains of the AA2060-T sheets, and a set of parameters for precisely describing the grain’s behaviour. The details of each feature are discussed in the following sub-sections.The present study used a dislocation-based single CP model as a constitutive equation of AA2060. The theoretical framework of the proposed CP model followed the previous works performed by Cheong et al. , Arsenli Fp part , where FDue to the incompressibility condition during the plastic deformation, the following conditions are required.L (velocity gradient tensor) is also decomposed to By similarity, L is described as follows:The velocity gradient tensor α-slip system, and n is the potential slip system’s number. At low homologous temperatures, it is assumed that the dislocation glide on well-defined crystallographic slip systems is the dominant source of plastic deformation. Thus, the plastic flow can be written as:ectively ,37,38. FIn addition, E is the elastic Green-Lagrange strain tensor defined as:Since the elastic deformations are infinitesimal compared with the plastic deformation, the linear Saint Venant relation can be used to describe the stress response of single crystals:rameters :(14)Cij=In the intermediated configuration, the resolved shear stress tensor The Orowan equation can be used to relate the crystallographic slip shearing rate with the collective motions of dislocations, as follows:Under a quasi-static condition, the average dislocation velocity can be approximated as:s et al.  proposedp and q are considered to be within the following range:For detecting the shapes of the profile of the energy barrier associated with the interaction between the obstacle and dislocation , p and qBy combining Equations (19)–(22), the evolution of the crystallographic shearing rates is expressed as:The evolutions of the total dislocation density are written as the competition of the storage and recovery mechanisms:Assuming the dominant source of the dislocation nucleation, the athermal storage rate can be written as:For the annihilation process, the cross slip of screw dislocations and the climb of edge dislocations are responsible for the dynamic recovery at low and high temperatures, respectively. According to Essman et al. , the scrThus, we further decompose the total dislocation density as:Therefore, the evolution of dislocation densities can be expressed as:−5.Since we are concerned with the deformation response at low homologous temperatures, the critical annihilation distance for edge dislocations is assumed to be constant for simplicity. Since cross slip is a thermally activated process, however, the critical annihilation distance for screw dislocations is also a function of temperature and stacking fault energy. Motivated by the form proposed by Nix et al. , we emplb is the Burgers vector’s magnitude, and λ is the statistics parameter to identify the deviations from the regular spatial arrangements of the dislocation densities. Additionally, According to the Taylor equation ,42, the dynamics ,44,45 ordynamics ,47. NeveCH is primarily based on the characteristics and the finite element (FE) modeling of the RVE, which represents the actual microstructure of AA2060. In the CH of metallic material, three models of RVEs were used. In the current study, a grain-based RVE model was utilized, in which every grain is represented by several cubic elements. Thus, further information about the size and the shapes of the grains in the metallic materials can be efficiently included. In this investigation, the established RVE model possessed equiaxed grains shapes. Furthermore, the model was discretized 100  figures might be reasonably obtained when the element numbers in the RVE model are acceptable. The main issue when establishing the grain-based RVE models is representing the initial textures of the received material. Most previous studies, for simplicity, assumed that all the materials had isotropic behaviours ,50,51. NThe mechanical behaviour of the RVE model was obtained by FE modelling, where the established model was used directly as FE meshes, in which each voxel describes one cubic finite element. Additionally, appropriate boundary conditions must be implemented on each face of the cubic element. Hazanov et al.  noticed To accurately predict the deformation behaviour of the AA2060 sheet utilized in this study under warm forming conditions by coupling CP modelling with CH framework, it was crucial to determine a set of material parameters that are necessary to properly describe the behaviour of grains and are utilized in the proposed dislocation density-based CP model. The set of parameters included The deformation behaviour of AA2060 sheets under warm forming conditions was predicted and captured by the FE modeling of the proposed RVE model. The grain’s behaviour for reflecting the actual deformation mechanisms was modelled via CP modelling. NMBERMSE, , AARE, and R, as listed in R is the correlation coefficient, which is a vital parameter calculated from Equation (33) to determine the strength of linear relations between predicted and experimental stresses. If the R nears 1, this signifies that the reliability of the proposed approach is outstanding [Further validation for the predictability of coupling CH with CP modeling was performed by calculating statistical parameters such as standing ,51.(33)N is the number of points utilized in this analysis. AARE) and root mean square error (RMSE), which are important for quantifying the capability of the proposed approach for predicting the warm deformation behaviour of the AA2060-T8 sheet correctly [AARE imply that coupling CH with CP modeling can determine that the warm reliability of the proposed approach is significant and vice versa [orrectly ,53,54. Tce versa .(34)AARNMBE), utilized to quantify the mean bias in predicted results. NMBE was calculated from Equation (36), where the positive value of NMBE means over-prediction; however, the negative value indicates under-prediction [The last statistical parameter used in this validation stage was normalized mean bias error (ediction .(36)NMB−1. For revealing the warm deformation behaviour of the AA2060 sheet, isothermal tensile testing was accomplished using a Gleeble-3800 thermomechanical simulator at temperatures and strain rates that varied from 373 to 573 K and 0.001 to 0.1 s−1. Then, a novel CP model was proposed for describing the grains’ behaviour and reflecting the crystals’ actual deformation mechanism under warm forming conditions. In the CP model, the total dislocation density was decomposed into edge and screw components to consider DRV caused by a cross slip of the screw dislocations at low homologous temperatures. The experimental stress-strain curves characterized the material parameters of the proposed CP model. After that, the proposed constitutive model was implemented using a monolithic time-integration algorithm-based backward Euler method. Afterward, to clarify the in-grain deformation and link the mechanical behaviour of AA2060-T8 with its microstructural state, RVE elements were created to represent the microstructure of AA2060-T8, where several finite elements discretized every grain. A remarkable accordance was observed between the predicted results and their experimental counterparts for all testing conditions. This signifies that coupling CH with CP modelling can determine the warm deformation behaviour of AA2060-T8  under different working conditions.In the current study, A new approach was proposed by coupling CH with CP modelling for predicting the warm deformation behaviour of AA2060 and


Due to its “generally recognized as safe status” (GRAS) and moderate treatment temperatures, non-thermal plasma (NTP) has lately been considered a suitable replacement for chemicals in the modification of food properties and for preserving food quality. One of the promising areas for the application of NTP is the treatment of wheat flour, leading to improved flour properties and product quality and consequently to higher customer satisfaction. In the present research, the German wheat flour type 550, equivalent to all-purpose flour, was treated using NTP in a rotational reactor to determine the influence of short treatment times (≤5 min) on the properties of flour , dough , and baking products . Based on the properties of NTP, it was expected that even very short treatment times would have a significant effect on the flour particles, which could positively affect the quality of the final baking product. Overall, the experimental analysis showed a positive effect of NTP treatment of wheat flour, e.g., decreased water activity value (<0.7), which is known to positively affect flour stability and product shelf life; dough stability increased (>8% after 5 min. treatment); dough extensibility increased (ca. 30% after 3 min treatment); etc. Regarding the baking product, further positive effects were detected, e.g., enhanced product volume (>9%), improved crumb whiteness/decreased crumb yellowness, softening of breadcrumb without a change in elasticity, and limited microorganism and enzymatic activity. Furthermore, no negative effects on the product quality were observed, even though further food quality tests are required. The presented experimental research confirms the overall positive influence of NTP treatment, even for very low treatment times, on wheat flour and its products. The presented findings are significant for the potential implementation of this technique on an industrial level. Plant-based food, such as cereals, grains, and their products, are an important source of energy for humans and animals due to their constituents, i.e., carbohydrates, proteins, vitamins, fats, and minerals . In the Food processing is a crucial step in the food supply chain of the modern food industry, which ensures food safety and intensifies food functional properties  ,3,4. TheModern lifestyles and the aspiration towards healthy eating have created a demand for minimally processed food, fewer synthetic additives, clean-label products, and highly natural ingredients . SustainWheat is ranked as the prime staple crop that ensures food and nutritional security . ChemicaIn the wheat dough formation process, gluten proteins are hydrated and are transformed into a continuous cohesive visco-elastic gluten protein network, accompanied by an increase in the extractability of gluten proteins . The funThe processing of wheat flour is a common practice on an industrial level, not only to improve its hygiene, safety, textural properties, and shelf life of products but also to affect the dough strength and weakening agents according to product specifications. In the food industry, chemicals such as ascorbic acid, azodicarbonamide (ADA), and potassium bromate are used as dough properties enhancers . For exa5 K) than of the gas bulk molecules (~300 K) [One promising approach to food treatment is the treatment with non-thermal plasma (NTP). Plasma, representing the fourth state of matter, is an overall neutral, ionized gas consisting of ozone in abundant amounts, along with other active species  ,46,47,48(~300 K) ,53. AtmoLately, NTP has gained much attention in food applications due to promising results in impacting quality attributes, food preservation, safety, and microbial inactivation ,55. The The goal of the present study is to investigate in detail the influence of the DBD type of NTP on wheat flour functionality and the properties of its products. NTP-type DBD was chosen due to its low, controllable temperature level and abundant amount of ozone production, along with other active species. To evaluate the potential of this method for an industrial application, very short treatment times  were applied. The analysis was based on evaluating the changes in properties of flour , dough , and baking product properties . Based on the detected positive and negative aspects of the NTP treatment, the applicability of this method for wheat flour treatment was discussed.In this research, Germany’s wheat flour type 550 , equivalent to all-purpose flour, was used for treatment, preparation of dough, and baking products (test bread). Flour, dough, and baked products were analyzed before and after the NTP treatment.The experimental setup , which wP = 120 W, f = 80 kHz, U = 7 kV) was used to generate the DBD plasma  made of stainless steel and mounted axially on a rotating shaft. To stop the formation of arcs, a dielectric barrier (thickness = 2 mm) made of Plexiglass was attached to the inner side of this ground electrode.(1) the outer (ground) electrode, in the form of a hollow cylinder  was attached to the high-voltage electrode to reduce the discharge gap to 2 mm and to reduce the overall power needed to generate DBD plasma inside the reactor. The uniform flour treatment inside the reactor is ensured by the presence of plastic baffles . Further details of the setup can be found in our previous publication [(2) the stationary, inner (high-voltage) electrode is positioned coaxially to the ground electrode. A row of aluminum pins (lication .Doughs were prepared from untreated and treated flour samples in order to identify and compare the changes induced by the NTP. Flour (3000 g), water , salt (60 g), dry yeast (75 g), fat (30 g), and sugar (30 g) were mixed in a dough-preparing machine . The kneading process included a mixing phase (at 25 Hz mixing frequency) of 2 min, followed by a kneading phase (at 50 Hz) for 6 min. After kneading, the dough was left to rest for 10 min, followed by a proofing step. The dough was divided into 6 parts weighing 600 g each. Three of them were left for normal proofing and the rest for extended proofing. Normal proofing was performed for 60 min (90 min in case of extended proofing) at 32 °C and 85% relative humidity. Afterward, the dough was used for a series of analyses.Six sandwich breads were prepared to observe the baking properties. They were baked free and settled on a baking plate  in an electric oven for 30 min. They were left to cool for 10 h at room temperature prior to baking analysis.Moisture content was determined three times per sample, according to the standard method (ICC Nr. 110/1) approved by the International Association for Cereal Science and Technology. Based on these measurements, the standard deviation was calculated.w-values) of the flour samples treated by NTP was obtained using the sorption isotherm method [The variation in the water activity (am method . The anaNear-infrared (NIR) spectroscopy is a rapid, accurate, and non-destructive technique that is commonly used in food analysis using the interaction between electromagnetic energy (in the range of 800–2500 nm) with the matter in question . It was In the present research, a standard method for determining gluten (ICC Nr. 155) was used to quantify the wet and dry gluten content and the gluten index (GI). The GI is correlated with the protein strength, which is calculated based on the percentage of wet gluten that remained on a sieve (84 microns for wheat flour) after centrifuging . The anaSedimentation analysis is a physicochemical test that provides information about the protein swelling capacity, which directly affects the baking quality  of a wheat flour dough. This analysis involves the absorption of water and the swelling of protein in a water-flour, lactic acid, and isopropyl alcohol slurry. The absorption capacity is dependent on the protein quality ; higher The adhesive properties of wheat flour were analyzed by kneading gluten balls ten times in a 2% indium, sodium chloride, and water solution for one hour.The farinograph analysis was performed according to the standard method (ICC Nr. 115/1). Dough quality is additionally characterized by dough stability. This is the ability of the shaped piece of leavened dough to maintain its shape during the proofing phase, which can be estimated by a farinograph. Stability is connected to the tolerance of flour to kneading and overmixing, and it is measured as the time during which a dough sustains its maximum consistency. It corresponds to the difference between the dough development time  and the departure time  on a farinograph chart. Dough softening is the difference between the heights of the farinograph curve between the point of the maximum resistance to mixing and the point measured 12 min afterward. Softening is concerned with the extensibility of dough. Flour that is too hard (higher protein content or S-H bonding) needs softening agents to break down the dough’s strength in order to meet the handling and similarity in baked products on an industrial level. The analysis was repeated two times, based on which the standard deviation was calculated.Dough’s resistance to stretch and gluten protein extensibility was analyzed by an apparatus, “Kieffer rig” . This apThe Falling number (FN) records the activity of α-amylase, which is responsible for the gelatinization and breakage of starch granules. α-amylase is an enzyme that breaks down a long-chain starch to simple sugars, which in the end, impacts the quality of baked products. The Perten-Hagberg number is the measure of the time (in seconds) required to travel a certain distance for a stirrer through a hot water-flour slurry . If the The gelatinization and pasting behavior of wheat flour can be studied by amylograph, which provides an easy and standardized performance and an effective correlation with the final product quality . The gelL*a*b color space that expresses color numerically and can be used for precise color communication [L coordinate represents the lightness , the a coordinate indicates the red to the green range, and the b coordinate indicates the yellowness. E* represents color uniformity, and ΔE shows the difference in colors. The analysis was repeated three times, based on which standard deviation was calculated.In 1976, the International Commission on Illumination (CIE) recommended a method CIE nication . The colChanges in starch and protein functional properties due to plasma treatment also affect the baking product properties. Starch and gluten interact to form a stable network that entraps the fermentation gas and resists a bread collapse during the baking and cooling stage . Starch To analyze the effects of NTP treatment on the baking product, the dough was prepared as presented in Wheat grains and flour can carry different contaminants from the environment as they are raw agricultural products. Microbial contaminants are mostly present on a grain surface, which are mostly removed during the milling process. However, flour can still contain dangerous contaminants . EnterobThe presence of the microorganisms sulfite-reducing clostridium, yeast, mold, E.coli, Salmonella, Lactobacillus, Staphylococcus, Bacillus cereus, and Enterobacteriaceae in flour was analyzed according to the German standard DIN ISO 21528-1/2.Before the treatment, the NTP reactor was thoroughly cleaned, and 150 g of type 550 flour was added to it. The reactor rotated at a constant speed of 10 RPM to ensure uniform flour-plasma contact. A high-voltage generator was operated at the maximum power (120 W). A maximum of 3000 g of plasma-treated flour was needed for flour, dough, and baked products analysis. Therefore, twenty batches of 150 g were treated in the batch reactor and mixed together in a vessel  before their use in the respective analysis.In order to investigate the effects of NTP on wheat constituents, the analysis was performed on untreated and treated wheat flour and corresponding wheat dough and wheat baked products. Following section lists and encloses the performed analysis in detail.Quantification of the amount of water is the first step in reporting the wheat quality attributes, especially the hygienic conditions. A variation in the amount of water within the wheat flour directly influences the flour’s stability and supports chemical reactions. Flour moisture content also affects its transportation properties, such as flow from silos, long-term transportation, compression, mixing, and packaging. Water content significantly affects the formation of liquid bridges and the capillary forces acting between the flour particles . This paMoisture content accounts for the bound and the free water present in flour. The dependency of the moisture content of wheat flour on the treatment time  is shown in p-value obtained by analysis of variance (ANOVA) is greater than 0.05  limits flour spoilage by microbial activity, also reported by Labuza et al. [The moisture content itself is not enough to predict the safety, stability, and shelf life of a product. The amount of free water available to support chemical or biological processes in flour, which determines the potential growth capability of microorganisms, is expressed by the water activity analysis in terms of the aa et al. .w-value remained in the optimal aw—range for the tested treatment time, thus ensuring the flour’s safety and stability during storage at room temperature. The ANOVA analysis (p < 0.05) , especially gluten protein , are important constituents of wheat flour that play a significant role in dough network development through the formation of disulfide bonds within protein molecules. They also have an important impact on the baking product quality due to their effect on the water absorption capacity, cohesivity, viscosity, and elasticity of a wheat dough . The folThe quantity and quality of gluten proteins are considered one of the major quality parameters for wheat flour. It has a major impact on the cooking quality, e.g., appearance, loss of solids in cooking water, and texture  . The effObtained results revealed that the wheat gluten content (dry and wet) of samples treated up to 3 min is comparable to the control sample, i.e., no significant change in the total protein level of wheat flour was noticed   ,88. Watep-value (>0.05) obtained by ANOVA analysis  obtained by ANOVA analysis  in wheat flour-dough protein properties is strongly related to the end product specifications. Thus, it is not easy to claim the extent of positive or negative change in protein properties due to NTP treatment. For example, due to the formation of –SH bonds, the dough is stronger, meaning that the handling of the dough is good and it can better retain its structure. On the other hand, loaf strength can negatively affect the volume of the baked bread while COStarch, which is a pure carbohydrate, represents the major part (63–72%) of the wheat grain consisting of glucose polymers, amylose, and amylopectin . Two typStarch quality and quantity directly affect the wheat dough’s strength, as starch is physically entrapped in a gluten network and acts as an inert filler during dough making . In bakep-value obtained by ANOVA analysis  for all the samples. The p > 0.05, provided in the Obtained results show no significant change . This confirmed no negative effect of the NTP treatment, i.e., the color of flour particles remained unchanged and uniform.L, b, a, and ΔE showed that plasma caused a lighter color in the baked wheat products.The CIE color coordinates values were also obtained for baked products made of untreated and NTP-treated flour and are summarized in In the presented research, the NTP effects on the baked product, i.e., bread volume, its softness, extension, and aroma properties, were investigated.p < 0.05)  the dough volume when the flour was treated with plasma. However, at the treatment time of 1 min, a maximum increase of 5% in volume was observed. This corresponds to the finding shown in 2 gas by yeasts.Studies ,93 indicp < 0.05, provided in the p > 0.05).The texture and pliability of bread are indicators of freshness and can be evaluated through texture analysis. Sensory analysis is vital for food products, as it establishes the quality and the acceptance of a product among defined target customer groups and markets. The sensory organs  are applied to analyze and evaluate the food product characteristics according to various schemes. DLG  is one of the oldest institutions in Germany that provides certified sensory tests for food and beverages . Ozone, p < 0.05, provided in the The studied flour was free from most of the detrimental microbial colonies; therefore, no reduction of the overall microorganism activity due to plasma treatment could be established. However, plasma treatment did result in a statistically significant reduction of Enterobacteriaceae , glucose oxidase (GOX)), and the NTP treatment on flour properties, the extensographic properties were emphasized rather than the farinographic. In this study, ASC and ASC + GOX were added (0.2%) to untreated flour samples to compare their enhancement properties with the potential of NTP treatment.Obtained results  depict tThis finding indicates that the proper NTP treatment of flour could significantly affect the flour’s extenographic properties and, in this sense, would be able to replace chemicals for this purpose. However, further investigation in terms of the optimal treatment time is needed.The overall conclusion, which can be drawn from the presented experimental analysis, is that the NTP treatment of wheat flour showed positive effects on flour properties and properties of its products, even for very short treatment times (≤5 min). Plasma species produced during NTP treatment showed a potential to affect the flour and dough structural network attributes, i.e., protein, starch, color, surface structure, enzymatic, and microorganisms activity to a certain extent. An important finding is that no negative effects on the product quality were observed.NTP treatment made treated flour slightly moister, which is expressed as an increase in the moisture content, but the amount of free water, expressed as the water activity, decreased. Decreased water activity (<0.7) is expected to result in improved flour stability and product shelf life. For the treatment duration of 5 min, flour protein strength (expressed through the GI) increased by 8%, which is an indicator of a more stable dough. The NTP treatment of 3 min resulted in an increased dough extensibility and resistance to elongation, i.e., an increase of >30% and >45%, respectively.Regarding a baking product, an increase in dough volume of, on average, 9% was observed for the loaves made from plasma-treated flour. This finding indicates the softening of a breadcrumb without causing a change in the bread elasticity. Even though the color of treated flour samples stayed unchanged, the whiteness of a breadcrumb was increased (>6%). The enzyme activity within flour decreased, which has a positive effect on safety; however, α amylase activity is reduced, which is essential for the dough’s softness. Longer treatment times (5 min) resulted in a decreased amount of fat in flour, which may reduce the flavor and mouth-feel properties of the baked goods.Presented results indicate that the NTP treatment of flour could be used as an alternative to chemicals for changing the flour and respective dough functional properties on an industrial scale. However, as an outlook to this work, the upscaling of this technology can be challenging in terms of the reactor design to provide a uniform, direct contact between flour and plasma species, as the optimal flour treatment procedure is vital for further implementation of NTP-based flour treatment. Even though some aspects of food safety were investigated, the applicability of NTP treatment in the food industry still needs to be approved, e.g., a review of the processes with regard to the Novel Food Regulation is suggested.


Linear ubiquitin chain assembly complex–assembled linear poly-Ub chains play key roles in immune signaling and suppression of cell death and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear poly-Ub via its Npl4 zinc finger (NZF) domain. Although the structure of the bound form of the HOIL-1L NZF domain with linear di-Ub is known, several aspects of the recognition specificity remain unexplained. Here, we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear di-Ub-bound state while rejecting other potential Ub species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear Ub binding, and interactions between the NZF core and tail revealed conserved electrostatic contacts, which were sensitive to charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear Ub affinity by weakening the integrity of the linear di-Ub–bound conformation. The described molecular determinants of linear di-Ub binding provide insight into the dynamic aspects of the Ub code and the NZF domain’s role in full-length HOIL-1L.Attachment of polyubiquitin (poly-Ub) chains to proteins is a major posttranslational modification in eukaryotes. Linear ubiquitin chain assembly complex, consisting of HOIP (HOIL-1-interacting protein), HOIL-1L (heme-oxidized IRP2 Ub ligase 1), and SHARPIN (Shank-associated RH domain–interacting protein), specifically synthesizes “head-to-tail” poly-Ub chains, which are linked  Although initial focus was on K48-linked poly-Ub chains and their role in shuttling target proteins to the proteasome for degradation, as time progressed, nonproteolytic roles of Ub chains increasingly emerged over time. Intriguingly, an unexpected eighth modification was discovered in 2006, in which Ub linked via the α-amino group of its N-terminal methionine residue (M1) instead ; however, its connection to immunological pathways and LUBAC function remains elusive  that has been shown to bind linear Ub chains avidly and that this interaction is important for suppression of NF-κB (itself has been shown to selectively bind to linear Ub chains via its NZF (Npl4 zinc finger) domain. This NZF domain contains a unique C-terminal region that is absent in other NZF domains—hereafter referred to as “NZF tail.” Both the core of the NZF domain and this tail bind to linear Ub, and it has been shown that disruption of this interaction attenuates NF-κB induction  K63-lin in 2009 ) remainsHere, we combined solution-state NMR spectroscopy, molecular dynamics (MD) simulations, isothermal titration calorimetry (ITC), and structural modeling to fill these gaps in understanding regarding HOIL-1L, which may also grant new insight in a broader context of poly-Ub recognition specificity, that is, the recognition dynamics of the Ub code .15N-isotope-labeled purified HOIL-1L NZF protein samples and conducted NMR titration experiments. In titration experiments using K63-linked di-Ub, we observed weak binding to the HOIL-1L NZF domain . For example, residues Cys197, Phe202, Ile203, and Glu212 of the NZF core  showed clearly detectable chemical shift changes upon increasing the di-Ub to NZF molar ratio. Three of the residues shown in Figure 1A, left  have previously  of approximately 224 μM  experiments and crystallographic data, it has been established that the (mouse) HOIL-1L NZF domain including the NZF tail (hereafter referred to as “HOIL-1L NZF”) selectively binds to M1-linked poly-Ub chains . HoweverF domain A, left. NZF core A, left seviously  been impy 224 μM . Thus, K218 gradually disappeared in the free state and then reappeared in the bound state , whereas Phe202, Ile203, and Glu212 showed gradual chemical shift changes albeit with marked line broadening over the course of the titration. As these two residues map to the distal Ub-binding site in the reported cocrystal structure, their involvement in distal Ub binding explains their large chemical shift perturbation  and line broadening (ΔR2) upon interaction with M1-linked di-Ub. Similarly, Thr207, a residue close to the proximal Ub-binding site, exhibited tremendous line broadening at intermediate molar ratios. For Thr207, interestingly at such molar ratios, multiple broad resonances are observable , indicating the presence of multiple intermediate states between the free and final bound states. The marked preference and high specificity in binding toward M1-linked di-Ub are also apparent in the side-chain indole amide resonance of Trp195, which albeit structurally buried is located close to the proximal Ub-binding site  or started to disappear in the free state to reappear in the bound state (indicating slow exchange). For instance, the signal corresponding to Argnd state A, right,ing site . The fulB), whereas M1-linked di-Ub specifically interacted with both regions. The NMR titration experiments are in fine agreement with the available crystallographic data, demonstrating specific binding toward M1-linked poly-Ub via both distal and proximal binding sites, whereas the K63-linked chain is only transiently bound by the NZF core and rapidly dissociates under fast-exchange kinetics .bound state, alone. When mapping the chemical shift changes observed in the NMR titration onto this crystal structure, indeed most interacting residues mapped to Ub-binding sites; however, a couple of residues demanded further explanation. For instance, the side chain of Arg235 of the NZF tail faces away from the binding sites of both Ub moieties; yet Arg235 displays significant chemical shift changes upon binding to M1-linked di-Ub. Likewise, the NZF tail residue Glu242 side chain points away from the proximal Ub moiety; in addition, even more distal  residues Glu243 and Arg246 of the NZF tail are not in contact with proximal Ub, whereas Gln250 is missing in the crystal structure and Gln247, Tyr248, and Gln249 are observed in multiple distinct conformers at different distances seen from proximal Ub (C); yet all these residues show significant chemical shift changes upon binding M1-linked di-Ub. In part, secondary chemical shift changes might be responsible for the CSP of these residues. Nevertheless, taken together, these observations suggested the possibility that the NZF domain undergoes some sort of an intriguing structural change upon binding that is not evident from the bound state cocrystal structure alone.Interestingly, not all chemical shift changes observed in the titration experiment could be unequivocally explained by the cocrystal structure of the HOIL-1L NZF/M1-linked di-Ub complex, in other words, by the structure of the ximal Ub C; yet alA. Elements of the NZF domain architecture, that is, the NZF core, tail, and core–tail linker region, are defined in Figure 2B, and side chains from key regions of the structure are highlighted.To gain insight into possible structural changes in the HOIL-1L NZF domain occurring upon linear poly-Ub binding, we set out to elucidate the yet-missing solution structure of the NZF domain in the free form. The resulting structural ensemble as determined by using solution-state NMR spectroscopy based on NOE-derived distance and residual dipolar coupling (RDC)-derived orientational restraints  and S4 iFigure 2C). For example, the HOIL-1L NZF backbone overlays well with the TRABID NZF1 domain, which has been reported in two different cocrystal structures with, respectively, K29- and K33-linked di-Ub , th1Z (2WX0 ), the NpJ3 (3B08 ). The NZD) than in the di-Ub-bound form. This might hint at the necessity for a conformational change during poly-Ub binding, that is, an outward flip of the NZF tail to grasp the proximal Ub moiety. Since the proximal Ub moiety is known to interact with both the NZF tail  and the NZF core  , which is devoid of recognizable secondary structure elements in both the free and bound forms and thus might possess a certain degree of backbone conformational flexibility. Indeed, the dihedral angles differed between the free and bound structures, with the largest change seen in the region Ser227–Gln229  , one sim7–Gln229 , consist7–Gln229 .232–Gln247), whereas the solution structure of the free form reveals a kink near the carboxyterminal end of this helix (A). Although a kink causing the helical curvature is not unusual in general   . Such chistal Ub . Taken t15N transverse spin relaxation rate measurements, a technique sensitive to nanosecond protein backbone motions. If two domains of unequal size in a multidomain protein undergo largely independent diffusion in solution, these domains experience different hydrodynamic friction so that different average R2 values are observed for these individual domains (A): the average relaxation rates for the NZF core and tail regions were, 7.9 s−1 and 6.7 s−1, respectively. This is close to the theoretically expected  in-phase 15N R2 value of 6.9 s−1 for the combined NZF (core–linker tail) domain  . This overall uniformity in the measured transverse relaxation rates indicated that both the NZF core and the tail diffused together in solution, consistent with the determined NMR structure (A).While these results indicated that the conformational flexibility of the NZF domain underwent changes during the interaction with (and thus appeared to play a role in binding to) linear Ub chains, the degree of conformational flexibility in the free NZF structure remained to be verified as it is not immediately evident from NMR structure determination alone. Therefore, to experimentally gauge the backbone conformational flexibility in the free NZF domain, we conducted  domains . Here, h domains A: the avtructure A.Figure 15N transverse relaxation rate (Rex) by R2 relaxation dispersion experiments showed an Rex <2 s−1 for all residues of the NZF domain, which is often used as a cutoff for R2 dispersion analysis. Thus, in the isolated NZF domain, no measurable micro-to-millisecond motion between the core and tail region appeared to take place, confirming the overall stable integrity of the NZF core–tail domain even in the absence of bound di-Ub.Because of the importance of both the NZF core and the tail in the function of the NZF domain, we also examined whether any micro-to-millisecond conformational exchange such as a hypothetical detachment of the tail from the core process took place in the isolated NZF domain. However, measurements of exchange contributions to the 15N–{1H} heteronuclear NOE (hereafter: hnNOE) measurements. This experiment can distinguish very rapid (hnNOE <0.2) from medium (hnNOE ∼0.5) and slow (hnNOE >0.7) backbone motions and thus reports on main-chain conformational flexibility in proteins on the pico-to-nanosecond timescale with higher values indicating conformational rigidity. The NZF core showed hnNOE values of approximately 0.7 , confirming that the NZF core is a rather rigid stably folded entity in solution. Interestingly, the region linking the NZF core and tail region showed only slightly lower hnNOE values , suggesting that this linker region is not fully flexible in solution despite the lack of secondary structure elements in this region. In the NZF tail region, the hnNOE values showed a gradual decline toward the C terminus , reflecting a much higher degree of conformational flexibility in the C-terminal half of the NZF tail helix. This residual local flexibility might confer the NZF tail with one of the unique characteristics of intrinsically disordered proteins, which have been shown to efficiently bind to target ligand molecules by being able to rapidly sample a large number of conformations in solution . In other words, the presence of bound M1-linked di-Ub resulted in stabilization of the conformation of the NZF tail. Although far C-terminally located residues such as Gln249 and Gln250 still displayed rather low hnNOE values, this behavior is consistent with the crystal structure in which these residues retain conformational flexibility even in the M1-di-Ub-bound form (C). Moreover, structural stabilization because of M1-linked di-Ub binding was also detectable in the NZF core, albeit to a lower extent. Taken together, backbone flexibility in the NZF domain differs between the free and M1-linked di-Ub-bound forms, with conformational stabilization taking place upon binding. Conversely, the residual conformational flexibility in the NZF tail of the free form might be important in the early stages of the binding interaction.To assess whether changes in the conformational flexibility of the NZF domain occur upon specific (M1) poly-Ub binding, we also conducted the hnNOE measurements in the presence of M1-linked di-Ub. Importantly, hnNOE values were markedly increased in the NZF tail C; red. Iund form C. MoreovA, multiple side chains of the NZF tail helix interacted with the surface of the NZF core. For example, the aliphatic side chain of Leu238 was found in close vicinity with the aliphatic part of the Thr207 side chain. Interestingly, not only NZF tail helix residues but also residues of the core–tail linker such as Tyr228 and Pro230 participated in interactions with the NZF core, an observation in line with the comparably high backbone stability observed for these linker residues . This is highlighted by Glu234, the side chain of which points toward a joint surface formed by Lys205 and Arg208. Likewise, Asp231 wraps around the surface formed by Lys205. Interestingly, two positively charged residues, Arg235 and Arg237, do participate in the interaction with the NZF tail, but mostly via the aliphatic part of their side chain, not the charged guanidinium groups, which were found to point outward from the structure. This indirect involvement of Arg235 in stabilizing the relative arrangement of the NZF core and tail fragments  also explains the large chemical shift change observed upon M1-linked poly-Ub binding for this residue (C) since at this location the helix orientation needs to change when going from a free to an M1-linked poly-Ub-bound state (D). While the interactions with the NZF core were numerous in the N-terminal part of the NZF tail helix, virtually no interactions with the NZF tail were found in its C-terminal part as the PISA web server . The NZF tail contains a total of seven negatively charged amino acids, resulting in a high local negative charge density that is only partially compensated by the presence of three arginine residues . As expected from the analysis of the core–tail interface , positively charged functional groups on the NZF core are positioned to interact with patches of negative charge density on the NZF tail . Specifically, the ε-amino group of Lys205 and the guanidinium group of Arg208 are electrostatically ideally positioned to interact with two patches of negative charge density each. Taken together, electrostatic interactions play a crucial role in stabilizing the relative arrangement of the NZF tail and NZF core.The electrostatic nature of the core–tail interaction was confirmed by calculating the electrostatic potential at the surface of the NZF tail  Fig. 4BBFig. 4B.C). The evolutionary conversation of residues important for proximal  and distal  Ub binding, as well as Zn2+ coordination residues, have already been discussed by Sato et al. , Glu231, and Glu234, were found to be highly conserved , whereas additional residues that introduce negative charge density into the NZF tail, Glu232 and Glu233, were at least partially conserved among species. In addition, this analysis revealed that the two key residues that specifically orient the NZF core to its tail, Tyr228 and Pro230, were also highly conserved indicating that a conserved YXP motif in the HOIL-1L NZF, where X provides an outward-facing polar side chain, is crucial for bringing about the correct orientation of NZF tail to the NZF core. Taken together, high conversation in sequence is not only important for preserving the zinc finger structure and the specific (linear) Ub-binding surfaces but also for stabilizing the internal core–linker–tail structural arrangement of the NZF domain itself.To further confirm the importance of these electrostatic interactions in stabilizing the HOIL-1L NZF domain, we compared the amino acid sequences of the HOIL-1L NZF domains from various species, ranging from humans and mice to chicken and fish C. The evo et al. . Interes207 (B), a residue sandwiched between the highly conserved residues Lys205, Pro206, and Arg208 (C), came into close proximity with a patch of high negative charge density centered around Glu234 of the NZF tail. Not only is Thr207 also highly conserved among species but also is the facing residue Glu234 as well (C). Thr207 has been identified as a residue that can undergo phosphorylation in full-length rat HOIL-1L in vivo  group into the core–tail interface.In the visualization of the electrostatic potential of the NZF tail, we made the intriguing observation that the side-chain hydroxyl group of Thr207 B, a resid Arg208 C, came i as well C. Thr207 in vivo  by the aversus wildtype HOIL-1L NZF. Consistent with published SPR data (A). ITC revealed an exothermic reaction (D) with a 1:1 stoichiometry, indicating that one NZF domain binds to one molecule of di-Ub. The dissociation constant was determined to be 5 μM, which agrees well with the observed slow exchange regime. Strikingly, the phosphomimetic mutation T207D decreased the affinity of the NZF domain for M1-linked di-Ub by about twofold (B). Although enthalpically binding was even more favorable than in the case of the wildtype NZF domain, a larger entropic penalty counteracted this effect, resulting in a dissociation constant of 10.3 μM (D). When Thr207 was substituted for Glu instead of Asp, the affinity toward M1-linked di-Ub was further decreased to about threefold (C). In addition, the stoichiometry decreased to 0.8 (D). This is likely because the longer side chain of Glu207 compared with Asp207 would bring it closer to Glu234, resulting in a larger Coulombic repulsion between these two patches of negative charge density. Such a partial disruption of the integrity of the NZF core–tail interface could also favor dissociation of the NZF–linear di-Ub complex. For instance, the structurally “outward-facing” NZF tail side chain of Glu233 contributes to di-Ub binding via salt-bridge formation with Lys63 of the proximal Ub moiety  residue Thr207 and (mutant) phosphomimetic Asp207. As anticipated from the inspection of the electrostatic potential . Conversely, for the T207D mutant, the distribution was overall shifted to larger core–tail distances and even some very large distances of >1.2 nm were observed . One example of such a frame is shown in Figure 5F, where the charge–charge repulsion appeared to have pushed Glu234 out of the initial NZF core–tail interface, destabilizing the helix structure and likely weakening interaction with proximal Ub. Although we do not claim here that these MD simulations perfectly retrace the actual motion occurring in solution, the relative difference between wildtype and phosphomimetic NZF under equivalent MD conditions establishes that the integrity of the NZF core–tail interface can be potentially disrupted by phosphorylation, thus also affecting Ub binding.To further solidify the link between the integrity of the NZF core–tail interactions, its disruption by an introduced negative charge density, and complexation with M1-linked di-Ub, we conducted a series of 100 ns MD simulations of the NZF–M1-linked di-Ub complex, that is, the bound state. This allowed us to sample multiple independent trajectories with different degrees of distal or proximal Ub motion relative to the NZF and then compare the distribution of the NZF core–tail distances as measured at the Cα atoms of Gluotential , the chatructure E; upper.observed E; lower.207) in the HOIL-1L NZF core can act as an important conformational switch that not only disrupts NZF core–tail interactions to displace the helix of the NZF tail but also weakens M1-linked poly-Ub binding affinity.In this study, we have solved the previously missing solution structure of the free HOIL-1L NZF domain by solution NMR spectroscopy. We have also demonstrated the importance of internal structural dynamics between the NZF core and NZF tail in the specific binding to M1-linked poly-Ub. Interestingly, we found that a previously reported phosphorylation site (ThrA), and as a result, the NZF tail did not easily detach from the core via millisecond dynamics  relaxation dispersion approach developed by Peter Wright’s group  from “correct” (“on-pathway”) binding (A). While this analysis is beyond the scope of the current study, it is likely that the observed weak binding to K63-linked di-Ub and mono-Ub , it consistently modeled the linker between the UBL and NZF domains as a long unfolded peptide in line with previously conducted domain identification  can bind to the same site on the E3 , thus providing a large central scaffolding helix that connects the N-terminal region -tagged mouse HOIL-1L NZF domain (residues Pro192—Gln250) encoded on a pGEX-6p1 vector was transformed into Escherichia coli–competent cells strain BL21 (DE3) using a vector available from previous studies (−1 ampicillin as selection pressure. Protein expression was induced in the early midlog phase (absorbance at 600 nm = 0.5) of the growth by the addition of 0.3 mM IPTG , and the zinc sulfate concentration was raised to 20 μM. At this point, the temperature was decreased to 15 °C, and the culture was continued for another 24 h before harvesting the culture by centrifugation ; the resulting pellets were stored at −80 °C.N-terminally glutathione- studies , 31. ThePellets were resuspended in lysis buffer consisting of 40 mM HEPES (pH 7.4), 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 1 μM zinc sulfate, and cell lysis was achieved by ultrasonication. The lysates were subsequently cleared by ultracentrifugation , and the soluble fraction was applied onto a glutathione sepharose 4 fast flow column . The column was washed with four column volumes (CVs) of lysis buffer, 2 CV of lysis buffer with an additional 500 mM sodium chloride, and 2 CV of lysis buffer with an additional 1 M of sodium chloride. At this point, the column was reequilibrated with lysis buffer, and the GST tag was cleaved by incubation with human rhinovirus 3C protease at 4 °C overnight. After proteolytic cleavage between the GST tag and the NZF domain, the NZF domain eluted from the glutathione resin, whereas any residual uncleaved GST-NZF would remain bound to the resin. The column was then washed with another 0.5 CV to elute additional NZF protein. Next, the solution was dialyzed against a buffer consisting of 50 mM Tris–HCl (pH 7.4), 40 mM sodium chloride, and 1 mM TCEP. After dialysis, the solution was applied to a HiTrap Q HP column  and subjected to ion exchange chromatography, using a sodium chloride gradient ranging from 40 to 300 mM. Finally, the NZF domain was purified by size-exclusion chromatography using a Superdex 75pg 16/60 column  in either gel filtration buffer  or ITC buffer . The final purity as assessed by SDS-PAGE Coomassie brilliant blue staining was 99%, and MALDI-TOF mass spectrometry confirmed the expected molecular weight.13C and 15N labeling, the HOIL-1L NZF culture was carried out under equivalent conditions using M9 minimal media harboring [U-13C] glucose and [15N] ammonium chloride (Cambridge Isotope Laboratories) as the sole carbon and nitrogen sources, respectively. Point mutation–carrying plasmids of the NZF domain (T207D and T207E) were created by polymerase chain reaction, and the mutant NZF proteins were expressed using autoinduction in N-5052 media at 20 °C for 60 h  equipped with a 5 mm 15N/13C/1H z-gradient triple resonance cryoprobe (Bruker BioSpin). Before NMR measurements, the NZF domain was buffer exchanged into NMR buffer  and placed in Shigemi tubes at a final sample volume of 300 μl. The concentration of the NZF domain for triple resonance experiments was 1 mM, whereas the starting concentration for the titration experiments was 0.1 mM. The details of the concentrations during the titration experiments are indicated in the figure legends. While we had reported the details of the triple resonance experiments employed for assignment of the NZF domain in its free and M1-linked di-Ub-bound forms before (15N-edited NOESY–HSQC (heteronuclear single quantum coherence) and 13C-edited NOESY–HSQC , the recycle delay was set to 5 s (∼10 T1 (15N)) to assure sufficient spin-lattice relaxation and thus restoration of equilibrium magnetization between acquisitions. Saturation of 1H was performed by a series of 120° 1H pulses during the recycle delay, that is, over a time of 5 s; saturation was turned off in the control experiment. The free NZF domain was measured at a concentration of 1.0 mM. The sample concentrations for measuring hnNOE values in the M1-linked di-Ub-bound form of the NZF were 0.5 mM NZF and 2 mM di-Ub, respectively. Two independent measurements were carried out to derive average values and the standard error of the mean. 15N relaxation dispersion measurements used the pulse sequence hsqcNr2rex3d employing a Carr–Purcell–Meiboom–Gill relaxation time of 50 ms with τCP values of 0, 25, 12.5, 8.33, 6.25, 4.17, 3.12, 2.08, 1.79, 1.56, 1.39, 1.04, 0.78, 0.61, and 0.50 ms. The pulse sequence is available under the URL http://www.moleng.kyoto-u.ac.jp/∼moleng_01/nmr/r2.html or upon request to the authors. Data points at τCP values of 0, 8.33, 1.04, and 0.50 ms were measured twice as a base for calculating experimental uncertainties. Transverse 15N relaxation measurements were conducted as previously described .In the hnNOE experiments  restrai12E5 PEG; Sigma–Aldrich) with 200 μl of NMR buffer  and 50 μl D2O and then stepwise adding small aliquots (1 μl) of hexanol (Sigma–Aldrich) with vigorous vortexing after each addition. The final amount of hexanol added was approximately 16 μl, at which point a clear solution was obtained. The PEG bicelle solution was then mixed 1:1 with protein solution, and the final solution was allowed to equilibrate in the NMR magnet for at least 1 h before measurements.To obtain the dataset of RDC constants, a PEG bicelle alignment medium was prepared by mixing 50 μl of pentaethylene glycol monododecyl ether (CDNH) and scalar coupling (JNH) values. Average values of the obtained DNH values were used in the subsequent analysis, and the uncertainties in DNH were estimated as the SEM among the two measurements.Two independent in-phase anti-phase-HSQC  experimeFindTensor.cya. For this determination, only a small number of RDCs was chosen, and the correlation coefficient for this initial tensor estimate was 0.99. For structure determination, the number of RDC restraints used was gradually increased (see the https://github.com/pinbo/boxshade).The alignment tensor was estimated using the initial CYANA structure (see aforementioned) independently in the program REDCAT  and CYANThe binding between linear di-Ub and the HOIL-1L NZF domain (wildtype or point mutant) was studied by ITC experiments at 298 K on a MicroCal iTC200 system . Both ligand and analyte proteins were dialyzed against ITC buffer overnight at 277 K and thoroughly degassed before each experiment. For most experiments, the concentration of linear di-Ub in the syringe was 1 mM, and the sample cell contained 100 μM of HOIL-1L NZF, whereas several experiments used half of these concentrations. A total of 19 injections of 2 μl each was carried out at 3 min intervals, and an initial 0.4 μl injection was discarded. The measured thermograms were processed using Origin 7 (OriginLab Corporation). Three independent experiments were conducted each, and the experimental data were fitted to a binary (“one set of sites”) binding model in Origin.3B08  were applied. To achieve adequate sampling of the conformational space, 20 independent 100 ns simulations were conducted, and analysis of trajectories was carried out using tools included in the GROMACS package.MD simulations were conducted in GROMACS, version 2021  under th3B08 ). Simula8IM5. All data are contained within this article and available from the corresponding authors on reasonable request.Atomic coordinates have been deposited with the RCSB PDB with the PDB ID code of This article contains The authors declare that they have no conflicts of interest with the contents of this article.