Lactic acid bacteria (LAB) have an extracellular proteolytic system that includes a multi-domain, cell envelope protease (CEP) with a subtilisin homologous protease domain. These CEPs have different proteolytic activities despite having similar protein sequences. Structural characterization has previously been limited to CEP homologs of dairy- and human-derived LAB strains, excluding CEPs of plant-derived LAB strains. CEP structures are a challenge to determine experimentally due to their large size and attachment to the cell envelope. This study aims to clarify the prevalence and structural diversity of CEPs by using the structure prediction software AlphaFold 2. Domain boundaries are clarified based on a comparative analysis of 21 three-dimensional structures, revealing novel domain architectures of CEP homologs that are not necessarily restricted to specific LAB species or ecological niches. The C-terminal flanking region of the protease domain is divided into fibronectin type-III-like domains with various structural traits. The analysis also emphasizes the existence of two distinct domains for cell envelope attachment that are preceded by an intrinsically disordered cell wall spanning domain. The domain variants and their combinations provide CEPs with different stability, proteolytic activity, and potentially adhesive properties, making CEPs targets for steering proteolytic activity with relevance for both food development and human health. Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae .,26.22,26PyMOL 2.5 by Scrödinger was used for protein structure analysis and visualization. The NetSurfP-3.0 tool was used for supplementary prediction of protein structural features including secondary structures and protein ID .Wg2. These homologies were supported by HHpred pairwise comparisons, predicting 100% homology probabilities based on E-values below 10−37  , but theEach of the 18 AF models were selected among the five ranked AF structure models. Usually, the best AF-ranked structure was selected, however, lower ranked AF models were used if the propeptide domain showed position errors lower than expected . The MSAThe PR-domain was defined as the S8 protease domain of subtilisin , whereasNFICC96H was characterized by the absence of both a W-domain and an attachment domain. Hereby, the W-domain was only observed in relation to the cell wall attachment domains. Some PrtP homologs differed from each other by the presence or absence of an inserted PA-domain. However, all PrtP homologs had a PP-domain region constituted by a prepeptide and a propeptide.The protein domain delimitation revealed 11 overall domain architectures of the PrtP homologous proteins, with subtilisin being the simplest, containing a PR-domain in addition to the N-terminal pre-pro (PP) peptide domain region . Within As most subtilisin-like proteases, PrtP homologs are synthesized as pre-proenzymes and first activated by autocatalytic cleavage of the PP-domain region after translocation across the cell membrane.NFICC96H  peptide for translocation over the cell envelope. SignalP did not identify a clear SS peptide for PrtPNFICC96H , though NFICC80, and PrtPNFICC200, which represented Clusters I-III, XI, and XII . The PR-domain of subtilisin differed from the other PR-domain by its smaller size of 274 aa and the absence of a tree-turned helix structure PR-domain  and ScpC (RMSD 1.6–18 Å) also indicated structural differences between the Fn-domains, but some Fn-domains were also suggested to share structure homologies   A. Howeveotif RGE . The Fn1MS22337 (RMSD < 3.2 Å)  B. The se binding . Fn2β4–β of ScpC . When thectively B. This sβ3–β4 and Fn3β5–β6 regions had different structures in the homologs. ScpA had no β-hairpin in Fn3β3–β4, but was the only homolog with a β-hairpin in Fn3β5–β6. Despite the distribution of the Fn3 domains in the phylogenetic tree, the other Fn3 domains showed structure homology when superimposed on the Fn3 domain of PrtPMS22337 (RMSD < 1.9 Å)  C. Fn3β5–MS22337  is located approximately 15 Å away from the catalytic cleft, whereas PrtPNFICC096H is the only homolog that lacks this C-terminal loop structure. The N-terminal loop region is shared among all the PrtP homologs and corresponds to the region  that is known to play a role in the substrate specificity of PrtP homologs from Cluster X [Wg2 and PrtPSK11 differ in two positions, and the swap of these aa residues results in the hybrid specificities of these proteases [β1–β2 is another loop region that points towards the catalytic cleft, and it has been suggested to entail a substrate-binding region in ScpA [β1–β2 shows structural variations among the homologs that may be related to their different substrate preferences. The major contribution to the substrate binding energy has recently been assigned to the Fn2 domain of ScpA, which recruits its substrate by long-ranged electrostatic interactions [Fn1 connects PR and Fn2 and keeps them in close contact, where loop regions of Fn2 can point towards the catalytic cleft. Fn2luster X . These Nroteases . This su in ScpA . This biractions . Complemractions , suggestThe interplay between the domains including PR, PA, and Fn2 seems to regulate the enzymatic activity of the PrtP homologs rather than single determining factors. The variable loop regions stand out as potential determining binding areas for substrate selectivity and specificity, whereas the core domain structures are conserved, ensuring the required conformational stability and flexibility for governing substrate selectivity and specificity.SK11 [SK11 [MS22337 and a casein micelle has been proposed, relying on the adhesive properties of the protease [The PrtP homologs contain different domains with adhesion potentials, indicating that PrtP homologs may mediate adhesion. PrtP homologs have been shown to have adhesive properties against polystyrene, mucin, and fibronectin ,60. The SK11 ,61. The SK11 . The str11 [SK11 . A modelprotease . The adhprotease . The divCarnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. In this present study, the PrtP homologs from the plant-derived bacteria are only theoretical proteins as their identification is based on genome data. The expression and activity of these proteases need to be validated to support that the PrtP homologs are present and biologically relevant in the LAB families Carnobacteriaceae, Enterococcaceae, and Leuconostocaceae. PrtP homologs are far from prevalent in all LAB strains [PrtP homologs are found in many different LAB species representing almost all LAB families, including  strains ,7, and p strains . TherefoLeuconostoc lactis, Leuconostoc mesenteroides, and Lacticaseibacillus rhamnosus strains isolated from berry, seashore plant, and human vaginal materials. PrtPNFICC80, PrtPNFICC200, and PrtR represent the diversity of Cluster XII, demonstrating that structurally similar proteases not only span different LAB species, but also span various natural LAB habitats.The PrtP homologs from plant-derived LAB strains are classified into six clusters , with Cluster V representing LAB strains from five different plant materials: berry, evergreen, flower, fruit, seashore plant, and tap/tuber root. In general, the clusters of PrtP homologs are from LAB strains isolated from various environments, indicating that certain domain architectures are not correlated with the environmental origin of the LAB strains. Cluster XII contains PrtP homologs from Leuconostoc mesenteroides strain NFICC96 and several L. helveticus strains. The four PrtP homologs of the L. helveticus strain CNRZ32 are divided into two phylogenetic clusters with PrtH in Cluster IX and the other homologs PrtH2, PrtH3, and PrtH4 in Cluster XI, corresponding to the previous subdivision of their PrtP encoding genes [L. helveticus has arisen by both gene duplication and horizontal gene transfer events [L. helveticus strains, as the plant-derived Leuconostoc mesenteroides strain NFICC96 potentially contains three PrtP homologs.The same LAB strain can contain structurally diverse PrtP homologs, as observed for the novel plant-derived ng genes . Phyloger events . HoweverS. thermophilus strain of soy milk, in which PrtSLMD-9 facilitates bacterial growth by a modest degradation of soy proteins [The similarity of the PrtP homologs among LAB isolates from different species and environments suggests that PrtP-encoded genes are shared among LAB strains via horizontal gene transfer. Hereby, the PrtP homologs may facilitate cross-over fermentations, in which a microbe of a traditional fermentation process is introduced to another protein substrate. This is supported by the introduction of the dairy-associated proteins . Extraceproteins . The actStructure modeling has been performed using AF, which has created a platform for the structural comparative analysis of PrtP homologs from diverse LAB strains. The delimitations of the protein domains have revealed more detailed domain architectures of PrtP homologs. The phylogenetic clusters of PrtP homologs have different domain architectures that do not necessarily correlate with the natural habitat or species of the bacterial strains. Horizontal gene transfer may explain the occurrence of similar PrtP homologs in different LAB species that are isolated from different ecological niches. The core folds of the structural domains are often evolutionarily conserved among the PrtP homologs, whereas variations are addressed to different loop regions. The PrtP homologs contain different combinations of fibronectin type-III-like domains. The Fn-domains could be divided into 12 structural groups, of which Fn1 and Fn2 would show the highest evolutionary conservation. Fn2 plays a role in substrate interaction together with other domains of the proteases. The functions of the other Fn-domains are more speculative, but are likely to contribute to the conformational stabilization of the PrtP homologs. The structure analysis of the domain boundaries has also emphasized the presence of an ID-characterized W-domain, preceding two different anchoring domains. The variable W-domains may mediate different protein interactions. Substrate interactions of PrtP homologs are complex, regulated by interactions between different domains. Domain–domain interactions may also affect the conformational changes of the PrtP homologs, with potentially different responses on environmental changes. This highlights the importance of defined domain boundaries in studying the impacts of different domains and domain combinations on the stability, proteolytic activity, and potential adhesive activity of PrtP homologs.


An analysis of the sensitivity of fault slip distance, fault dip angle, and fault position to the maximum deformation of retaining piles was conducted. It was determined that the fault dip angle has the highest sensitivity, followed by fault slip distance, while fault position has the lowest sensitivity. Based on the fitting of 64 sets of orthogonal experimental data, a good linear relationship was established between the maximum deformation of retaining piles (Uhm) and the indicator η(The mechanical properties of soil in fault-fracture zone areas are diverse and complex. Deep excavation projects often encounter adverse geological conditions such as reverse faulting, which can lead to surface subsidence and collapses, posing significant challenges to excavation safety. Currently, there is limited research in the field of deep excavation engineering that analyzes the influence of reverse faulting on the deformation of retaining piles, and the existing research methods are not systematic enough. Therefore, this study aims to investigate the characteristics of how reverse faulting affects the deformation of retaining piles in deep excavation projects. Various research methods were employed, including numerical simulation, on-site monitoring, and orthogonal experiments, using a deep excavation project in Shenzhen as a case study. The results of the study indicate that reverse faulting exacerbates the deformation of retaining piles, causing the trend of increased deformation to shift upward. The upper part of the pile is significantly more affected than the lower part, and the overall deformation of the pile exhibits an approximate spoon-shaped curve distribution, with the maximum deformation occurring in the upper-middle section of the excavation. Under the influence of reverse faulting, the deformation of retaining piles is positively correlated with fault slip distance and fault dip angle, while it is negatively correlated with fault position. The growth rate of the maximum deformation of retaining piles, denoted as  The existence of fault slippage not only causes damage to engineering structures such as cross-fault buildings and underground engineering structures, but also poses a serious threat to the structural engineering in its affected area9. Excavation engineering located in the influence zone of a fault zone is easily affected by fault activity, leading to a series of adverse consequences such as displacement of the excavation support structure, uplift and cracking of the bottom plate, and damage to anti-seepage structural facilities. Therefore, fault activity poses a great threat to the safety of excavation engineering in its affected area. It is necessary to conduct research on the impact of fault activity on the deformation of excavation support piles to address this situation.As urban construction continues to develop, the utilization of underground space has attracted widespread attention. In urban engineering construction, the existence of special geological environments, such as active fault zones and fault slippage, has caused serious damage and threats to engineering10, in the context of a circular foundation pit excavation support project in the sandy soil layer of the West Anchorage of Humen Second Bridge, proposed a novel method for parameter calibration based on continuous wall displacement monitoring data. They also developed modeling and computational theories for the excavation support process of the circular foundation pit. This allowed them to obtain insights into the stress-deformation mechanisms of the soil layers and the supporting structures during the excavation and support of the sandy soil layer in the circular foundation pit.Zhang et al.11 proposed a calculation method for the deformation of retaining walls considering the spatiotemporal effects. The method was based on theoretical analysis, numerical calculations, and field measurements. The researchers used the influence coefficient of pit corner effect and the equivalent horizontal resistance coefficient to measure the impact of spatiotemporal effects on support deformation. Qin et al.12 mainly studied the influence of the reinforced area within the deep silt layer pit, the embedding depth of support piles, and the stiffness of support piles on the deformation law of foundation pits, and proposed corresponding deformation control measures. Mei et al.13, through statistical analysis of field measurement data from the excavation of ten subway stations in Xi'an, investigated the spatiotemporal characteristics of surface settlement and pile lateral deformation caused by deep excavation in expansive loess. Furthermore, they delved into the deformation mechanisms of excavations in loess regions, which is of significant importance for enhancing the design theory of excavations in this area. Zhang et al.14, in response to the safety requirements for deep excavation construction imposed by the construction of large-span high-speed railway arch bridges, based on the Xu-Honghe River Special Bridge Project of the Xuyan High-speed Railway, employed numerical simulation methods and combined them with on-site measurement data. They conducted a systematic study on the deformation and stress characteristics of steel sheet piles during the construction of deep excavation support structures for seismic fault zones in large-span railway arch bridge projects. Lei et al.15 conducted a numerical simulation of fault slip instability under the influence of horizontal structural stress using the 21,221 working face in the Yima Coalfield as the engineering background. They studied the evolution characteristics of the displacement field of a fault structure with a dip angle of 75° under mining disturbance.Baziar et al.16 studied the interaction between shallow foundations and reinforced soil under normal and reverse faults, and verified the accuracy of numerical simulation results through experiments conducted at the University of Dundee and Waseda University. Liu et al.19 studied the deformation of the strata, tunnel strain, and failure characteristics caused by a 75° reverse fault through model tests. Based on this, the force and deformation mechanism of a tunnel under the influence of sliding along a 75° dipping normal fault was studied through indoor model tests. Subsequently, the finite element method was used to compare and study the influence of different lining section shapes on the structural force and deformation of the tunnel under fault sliding. Triantafyllaki et al.20 achieved important research results in numerical analysis of the response of deep-sea buried pipeline structures crossing active normal and reverse faults. Jin et al.21, based on a deep excavation project in a karst and fractured zone formation near a subway station in Nanjing, comprehensively analyzed issues related to insufficient embedment depth of the ground-anchored wall, which led to sudden water inflow at the pit bottom and pit deformation. Subsequently, they conducted optimization design of the protective structure and implemented measures for controlling the water inflow. Post-assessment results demonstrated significant improvements resulting from these measures.Liu et al.22, through the statistical analysis of 42 engineering cases of foundation pit projects, investigated the influence of significant factors on the vertical and horizontal displacements of tunnels. They proposed a comprehensive prediction index for the horizontal displacement of tunnels and presented empirical prediction formulas for tunnel horizontal displacement under three different geological conditions. Xue23 based his research on a deep foundation pit project at a station on Changchun Metro Line 5. Using time series, he obtained training data and integrated the GA algorithm into the PSO algorithm for parameter optimization. By combining grey theory, he established a deformation time series prediction model for the foundation pits based on Grey Least Squares Support Vector Machine (Grey-LSSVM).By analyzing the research of domestic and foreign scholars on foundation pit engineering and similar projects, this paper further consolidates the research foundation. Gao et al.In summary, through the analysis of research findings from multiple scholars in the field of foundation pit engineering and fault activities, it can be observed that there is currently limited research in the field of foundation pit engineering on the impact of faults on the deformation of retaining piles, and even less research analyzing the influence of reverse faulting on the deformation of retaining piles. Therefore, conducting research in this area holds significant importance for both theoretical understanding and practical engineering applications. This study focuses on a specific foundation pit project in Shenzhen City. By comparing and analyzing numerical simulations and field measurements of the foundation pit retaining piles, the reliability of finite element calculations is validated. The study specifically emphasizes the investigation of the deformation effects of the foundation pit retaining piles in the influence zone of the footwall affected by reverse faults. The main research content includes analyzing the deformation characteristics of the retaining piles under the influence of reverse fault, considering different fault slip amounts, dip angles, and positions, and assessing the sensitivity of these parameters to the maximum deformation of the retaining piles. Finally, through fitting 64 sets of orthogonal experimental data, a prediction model and a project-specific prediction formula for the maximum deformation of the foundation pit retaining piles in the influence zone of the footwall affected by reverse fault are determined.24. The safety level of the excavation support system is classified as Level 1. The total area of the foundation pit is approximately 8900 m2, with a perimeter of about 380 m. The maximum excavation depth is 16.8 m. The support structure consists of piles and internal bracing, with two rows of internal bracing at a spacing of 8.5 m. The diameter of the retaining piles and column piles is both 1.2 m, with a length of 25.8 m for the retaining piles and 20.0 m for the column piles. The cross-sectional dimensions of the crown beam, waist beam, and support beams are all 1.0 m × 1.2 m. The concrete strength grade for the retaining piles, support beams, connecting beams, and column piles is C30. The cross-sectional dimensions and physical–mechanical parameters of the support structure are shown in Table This project is located on the southeast side of Yijing Station, Line 5 of the Shenzhen Metro, in the Luohu District of Shenzhen. The plan view of the foundation pit is shown in Fig. 1The site is located within the South China Series Bijiashan Formation (NhB) and is influenced by the F1322 Fault Zone (Shehe Cang-Henggang-Luohu Fault Zone). The fault zone is the main fault within the fault system, with a length of 38 km and a width ranging from 7 to 70 m, reaching a maximum width of approximately 267 m. It strikes in the range of NW 25°–55°, dips towards the southeast at an angle of 60°–80°, and is a high-angle reverse fault active during the Holocene. Due to tectonic movements at different periods, the fill materials in the fault zone include clay minerals, talc, quartz, etc., with relatively poor mechanical properties, and the fault surfaces are rough.24. The simplified stratigraphic structure and thickness of the site from top to bottom are as follows: (1) Uncompacted fill soil with a thickness of 4.4 m; (2) Clay with a thickness of 3.05 m; (3) Angular gravel with a thickness of 0.8 m; (4) Sandy clay with a thickness of 4.6 m; (5) Completely weathered rock with a thickness of 6.95 m; (6) Strongly weathered mixed rocks with a thickness of 6.90 m; (7) Moderately weathered mixed rocks, which have not been penetrated. Affected by the F1322 Fault, the bedrock at the site is relatively fragmented, and local occurrences of green clay alteration can be observed. The stability of the area where the site is located is relatively good, belonging to a stable region.The soil parameters selected based on the investigation report are listed in Table 2The MIDAS GTS NX finite element analysis software was used to establish a three-dimensional numerical calculation model. In order to reduce boundary effects and fully reflect the fault slip phenomenon, the numerical calculation model size was selected as length × width × height = 320 m × 220 m × 45 m, as shown in Fig. In order to accurately depict the water cutoff effect of the interlocking piles and cutoff curtain, the MIDAS GTS NX software was used to simulate the cutoff curtain using interface contact elements, with a permeability coefficient set to 0. For computational simplicity, the fault plane was assumed to have a regular geometric shape and was also simulated using the interface contact elements in the MIDAS GTS NX software. The interface element parameters were automatically calculated by the finite element interface assistant based on the parameters of the rock and soil on both sides of the contact plane, and the comprehensive judgment resulted in the interface calculation parameters shown in Table Before conducting finite element dynamic simulation calculations, horizontal constraints were applied around the model, and vertical constraints were applied at the bottom. By fixing the free movement of the lower part of the fault, a certain forced displacement is applied to the upper part of the fault to analyze the deformation effects of the foundation pit retaining piles in the influence zone of the footwall affected by the reverse fault. Similarly, by applying appropriate constraints to the corresponding boundaries, the deformation effects of different fault dip angles and fault positions on the retaining piles were simulated under certain slip amount. The initial fault parameters were set as follows: fault distance from the excavation is 25 m, fault dip angle is 72°, and fault amount is 9 mm. The boundary constraints and loading conditions for this simulation are shown in Fig. Through finite element calculation, the horizontal displacement cloud maps of the retaining piles under completion of excavation and bonding slip along the reverse fault are obtained, as shown in Figs. Extracting the calculation results of the horizontal displacement of a point (JCX5) on the side of the retaining pile near the fault, we obtain the deformation curve of the retaining pile, as shown in Fig. When some bonding slip occurs along the reverse fault, the retaining pile undergoes further deformation due to the compressive action between the fault surfaces. The trend of increased deformation shifts towards the upper part, and the effect of deformation is more significant in the upper part of the pile. The maximum deformation occurs near the middle-upper part of the pit, and the overall deformation of the retaining pile exhibits an approximate spoon-shaped distribution. At this stage, the maximum horizontal displacement of the retaining pile is 7.15 mm.The on-site measurements for this project include the deep-seated horizontal displacement of the retaining piles. Monitoring points were arranged with one monitoring point every five interlocking piles, totaling approximately 43 monitoring points, as shown in Fig. JCX5) were extracted, and a comparative analysis was conducted with the numerical calculation results, as shown in Fig. The measured results of the horizontal displacement of the pile body at the monitoring point near the fault side of the excavation pit , it was found that the maximum deformation growth rate of the retaining pile increased approximately logarithmically with the increase of fault slip amount.To further quantify the results, the maximum deformation values of the retaining pile under different slip amounts were extracted, and the curve of the maximum deformation of the retaining pile was obtained, as shown in Fig. Keeping the fault slip amount and position unchanged, the fault dip angle was adjusted in sequence to 45°, 62°, 72°, 82°, and 90° to study the deformation characteristics of the retaining piles with the increase of fault dip angle. The deformation impact law curves of the retaining piles under different fault dip angles were obtained through finite element calculations, as shown in Fig. r2 of the retaining pile was introduced, and it was found that the maximum deformation growth rate of the retaining pile approximately follows an exponential function with the increase of fault dip angle. Therefore, even with a small fault slip in a high dip angle reverse fault, it still has a significant impact on the deformation of the retaining pile.To further quantify the results, the maximum deformation values of the retaining pile under different fault dip angles were extracted, and the maximum deformation impact law curve of the retaining pile was obtained, as shown in Fig. Keeping the fault slip amount and dip angle unchanged, the distance between the fault and the foundation pit was sequentially adjusted to 15 m, 20 m, 25 m, and 30 m to study the deformation characteristics of the retaining piles as the fault distance from the pit increased. Through finite element calculation, the deformation influence curves of the retaining piles under different fault positions were obtained, as shown in Fig. r3 of the retaining pile was introduced. It was found that the maximum deformation growth rate of the retaining pile decreased approximately logarithmically with the increase of the fault distance from the pit, and when the fault distance from the pit reached a certain distance, the impact of this fault was very small.To further quantify the impact, the maximum deformation values of the retaining pile were extracted at different fault positions, and the maximum deformation influence rule curve of the retaining pile was obtained, as shown in Fig. Y being determined by n independent variables X1, X2, … , Xn. The function relationship between Y and X can be expressed as: Y = f. Under a certain condition, the independent variables are set as reference variables X', resulting in a reference dependent variable Y' = f. The analysis involves examining the deviation degree and trend of the dependent variable relative to the reference dependent variable, known as sensitivity analysis25.Assuming a set of variables, with the dependent variable Pi (Xi) is defined as:In order to compare the sensitivity of multiple independent variables, non-dimensional sensitivity functions and sensitivity factors are defined. The sensitivity function X'i into Eq. , increases approximately exponentially with an increase in the fault slip amount and dip angle, and decreases approximately logarithmically with an increase in the distance from the fault to the pit.The deformation of retaining piles under the influence of a reverse fault is directly proportional to the fault slip amount and fault dip angle, and inversely proportional to the distance between the fault and the foundation pit. The variation rate of the maximum deformation of the pile body, Sensitivity analysis of the maximum pile deformation to three parameters—fault slip amount, fault dip angle, and fault position—revealed that the fault dip angle had the highest sensitivity, followed by the fault slip amount, while the influence of the fault position was the smallest.Uhm had a good linear relationship with the indicator η(Through fitting the data from 64 sets of orthogonal experiments, it was found that the maximum pile deformation This paper, based on a deep excavation project in Shenzhen, utilizes the MIDAS GTS NX finite element analysis software to investigate the influence of reverse faulting on the deformation of foundation pit retaining piles. The following conclusions have been drawn:These research findings can serve as a valuable reference for the study of foundation pit engineering under similar geological conditions. Future research can be expanded to include various foundation pit projects under different geological conditions, thereby broadening the scope of the study and validating the applicability of the research results. Additionally, further exploration of the influence of different numbers and positions of faults on pile deformations can enhance our understanding of the complex interactions between faults and retaining structures.


Three-dimensional covalent organic frameworks possess hierarchical nanopores, enormous surface areas with high porosity, and open positions. The synthesis of large crystals of three-dimensional covalent organic frameworks is a challenge, since different structures are generated during the synthesis. Presently, their synthesis with new topologies for promising applications has been developed by the use of building units with varied geometries. Covalent organic frameworks have multiple applications: chemical sensing, fabrication of electronic devices, heterogeneous catalysts, etc. We have presented the techniques for the synthesis of three-dimensional covalent organic frameworks, their properties, and their potential applications in this review. Covalent organic frameworks (COFs) are two- or three-dimensional 2D, 3D) families of materials, which, through interactions between organic precursors, produce strong, covalent networks that enable the formation of porous, constant, and crystalline solids ,2. COFs D, 3D famFunctional groups are incorporated during the synthesis of COFs. These functional groups can be incorporated through two different methods: 2D layered structures  and 3D eThe stacked layers are held together through noncovalent interactions, which impart a large intrinsic freedom to the layers. Two orthogonal phase spaces are used for classifying this freedom: the interlayer distance and the layer offset. The presence of a zero offset vector indicates the formation of eclipsed stacking (AA) between the consecutive layers. The presence of a nominal value of non-zero offset indicates slipped AA stacking. The slipped AA stacking forms a type of serrated stacking called AB stacking. The vertices of the layer lie above the pore centers of the consecutive layer to form the AB stacking.On the other hand, 3D COFs form a framework with open sites and are bonded through strong covalent bonds. The functional groups in 3D COFs and 2D COFs align differently, resulting in different interaction patterns ,9,10. ThMetal–organic frameworks (MOFs), 3D porous coordination polymers, are formed by secondary building blocks, such as organic ligands and metal ions and clusters. COFs and MOFs are both porous coordination polymers, which make them attractive candidates for various applications. They have porous volumes and π-conjugated bonds. An accurate choice of linkers can impart tunable properties. The bonds are concurrently formed and broken with organic linkers during the formation of a MOF structure which helps in the growth of good crystals. The secondary building blocks in MOFs can alter their structure and size, keeping the topology intact. The presence of long organic linkers makes them highly porous. In contrast, covalent bonds are formed rigorously in COFs, leading to the formation of poor crystalline structures ,11. ThusThe substitution of rigid building units with flexible building units imparts flexibility with significant characteristics in the presence of external stimuli . NeverthIt has been a challenge for the synthesis of 3D COFs to integrate with flexible building units, due to the absence of additional weak interactions between the interlayers . The fleAlthough there are few reviews on the application and project of 3D COFs ,16 and tstp topology, low density, large pores, and a high surface area. The presence of wide channels within the crystal aided in the uptake of a large protein molecule, myoglobin, as shown in 2,3,6,7,14,15-hexakis(4′-formylphenyl)triptycene (HFPTP), a building unit with six connections, was used to design a 3D COF (3D COF-triptycene) . The crystp topology [Two triptycene-based COFs  were synthesized by [6 + 4] a condensation reaction of imine, as shown in topology . Nanocha2(TAP)], an amine with 4 connections, were allowed to undergo [3 + 4] condensation to form a 3D COF with a tbo topology having a non-interpenetrated porous skeleton, as shown in dT-octahedron. The linkers were placed on the triangular faces of the truncated octahedron alternately. There were 3 types of polyhedral cages in this topology. The COF is a good photocatalyst for the removal of fluorine from trifluoromethyl aromatics and the conversion of phenylboronic acids to phenols. The photocatalytic reaction took place both on the surface of the COF and within the crystal.4′,4‴,4‴″-nitrilotris[-4-carbaldehyde] (NBC), a trigonal aldehyde with 3 connections, and 5,10,15,20-tetra(4-aminophenyl)porphyrin  condensation reaction with square precursors (2D-Ctopology . However2(BF4)] was exploited by using it as a building block that was combined with benzidine to synthesize the imine threads, as shown in dia topology. The topology and architecture did not change after removing the metal ion. However, on remetalation with Cu(I), the complete structure was refurbished reversibly. The aldehyde groups were present in the para positions of both the phenyl substituents. A distorted tetrahedral geometry was formed where the two planes of phenanthroline form a dihedral angle of 57° with Cu(I) at the center. The π−π interaction between the neighboring planes of phenyl units and phenanthroline distorted the tetrahedral geometry  were placed perpendicular to each other by the spiroborate linkers. The presence of minute cavities in the skeleton imparted rigidity to the structure. Moreover, the interpenetration was hindered by the short linker. The topologies with their expanded form are shown in Spiroborate linkers with negatively charged sp2, a building block with the shape of a triangular prism, was used to design a 3D Cage-COF using 2,5-dihydroxyterephthalaldehyde (DHTPA) as the linear linker  in 2D-2hD (a pyrene-based monomer) and 3D-3hD (a triptycene-based building block) formed the 3D COF-triptycene. The difference between the surface areas of Ni-DBA-3D-COF and the triptycene-based COF is considerably large. The crystal showed two different pore sizes of 41 Å along the 2 g−1) with a non-interpenetrated dia topology. However, the size of the pores (26.5 Å) is comparatively smaller. These COFs were modulated by altering the methoxy substituents of the tetrahedral building blocks based on the concept of steric hindrance. A highly substituted linker imparted structural rigidity and reduced the dynamic nature of the skeleton. In a similar line, the surface area of 3D COF-PI-1 having polyimide as a monomer has a surface area of 2403 m2 g−1. The crystal is created with a combination of both non-interpenetrated and four-fold penetrated dia topology. Another 3D COF, 3D-COF-FPBA, with a ctn topology also has a noticeably high surface area and large pore size (13.6 Å). JUC 552 is a 3D meso-COF with the next highest surface area  was fabricated by coating a fluorescent aggregation-induced emission-based 3D COF on a blue LED bulb for emitting white light, which paved the way for white light-emitting diodes devoid of rare-earth metals, as displayed in A 3D COF having two different linkers, boroxine and imine, showed good bifunctional catalytic properties . The imiAnother pyridyl-based 3D COF was used as a heterogeneous catalyst for another Knoevenagel condensation reaction, as shown in An Ni-dehydrobenzoannulene-based 3D COF can be used to catalyze the polymerization of ethylene . A Pd(II)-spirobifluorene-based 3D COF catalyzed the Suzuki–Miyaura reaction between phenylboronic acid and aryl halide, as shown in An Ni-dehydrobenzoannulene-based 3D COF effectively separated ethane and ethylene from a mixture . The upt2 capture under mild conditions after six runs (yield > 90% and perfect turnover number), as shown in An azine-linked 3D COF was prepared with high porosity and excellent thermal stability, which illustrated good performance for COAnother imine-based 3D COF demonstrated the uptake of tetrahydrofuran, cyclohexane, and 1,4-dioxane as the adsorption characteristics were dependent on the guest molecule, as shown in 1,4-phthalaldehyde-derived 3D COF is highly porous and exhibited an iodine uptake capacity of 82.4 wt% at room temperature . A chargRs = 1.52) [S-enantiomer was obtained initially, followed by the R-enantiomer. A combination of channels resembling chiral tubes and amphiphilic tubes was present in the 3D COF. The inner part of the channels was covered by a layer of chiral hydroxyl groups. These chiral hydroxyl groups interacted with the guest alcohol groups and caused selective adsorption. The COF-based column could not separate a racemic mixture indicating, its potential for the identification of chirality.An enantiopure tera-aldehyde-based chiral 3D COF was used as the packing material of columns and was used for the resolution of stereoisomers, 1-phenyl-2-propanol with a good chromatographic resolution ( = 1.52) ,59,60. TAnother salen-based 3D COF was used as a stationary phase in high-pressure liquid chromatography (HPLC) columns for the efficient separation of ethylbenzene and xylene isomers, as shown in 3NH3PbI3 film reduced the leakage and showed good diode properties.The conjugation in spirobifluorene-based COFs was used for harvesting solar energy by doping photoelectric devices with the COFs . SpirobiSBFdiyne-based 3D COF films doped with tetracyanoquinodimethane (TCNQ) showed a high conductivity due to the existence of orthogonally placed conjugated polymers in the COF ,63, as s2•−) [2•− is catalyzed by redox-active metal centers in salens. The chemical stability, biocompatibility, and large surface area are effective at removing the oxygen radical. The redox-active metal center is responsible for the catalytic activity. However, a high concentration of the catalyst is required for the removal of oxygen radicals.Cu(II) containing 3D salphen-based COFs eliminated superoxide radical anion (O2•−) , which iThe review summarizes the synthesis and potential application of different 3D COFs. The major limitations of 3D COFs are fewer building blocks and topologies. Nonplanar building blocks with suitable functional groups are still limited to be applied as nodes in the synthesis of 3D COFs. Since designing a topology is the basic criterion for creating new 3D COFs, new building units should be further investigated.The functionalization of 3D COFs is significant for different applications. Furthermore, 3D COFs are functionalized at four different points: groups introduced during the latter part of synthesis, building blocks, linkages, and guest molecules. The groups introduced later can destroy the crystal structure. However, crystallization problems can be thwarted by introducing functional groups at the later part of the synthesis process. Building blocks and linkages are uniformly distributed throughout the framework. However, there are limited suitable linkages and functional groups as inappropriate functional groups tend to destroy the crystallinity of 3D COFs.The recyclability and stability of guest molecules are low. Thus, the functionalization of 3D COFs requires an extensive study to synthesize new 3D COFs using new functional groups. Additionally, both 2D and 3D COFs can be prepared utilizing the same functional groups and their diverse properties can be explored.Although 3D COFs have been explored as heterogeneous catalysts, the storing of gas and their separation, chemical sensing, fluorescence, and absorbing materials, there is a huge scope for their applications in diverse areas that are yet to be investigated. Design methods are required to obtain non-interpenetrated structures having large pore sizes and surface areas for further applications.


Cyclic-nucleotide binding (CNB) domains are structurally and evolutionarily conserved signaling modules that regulate proteins with diverse folds and functions. Despite a wealth of structural information, the mechanisms by which CNB domains couple cyclic-nucleotide binding to conformational changes involved in signal transduction remain unknown. Here we combined single-molecule and computational approaches to investigate the conformation and folding energetics of the two CNB domains of the regulatory subunit of protein kinase A (PKA). We found that the CNB domains exhibit different conformational and folding signatures in the apo state, when bound to cAMP, or when bound to the PKA catalytic subunit, underscoring their ability to adapt to different binding partners. Moreover, we show while the two CNB domains have near-identical structures, their thermodynamic coupling signatures are divergent, leading to distinct cAMP responses and differential mutational effects. Specifically, we demonstrate mutation W260A exerts local and allosteric effects that impact multiple steps of the PKA activation cycle. Taken together, these results highlight the complex interplay between folding energetics, conformational dynamics, and thermodynamic signatures that underlies structurally conserved signaling modules in response to ligand binding and mutational effects. A, inset). Despite high-resolution structures of CNB domains have been available for decades, there remains a lack of understanding in how CNB domains enable the CNB signal to regulate a diverse array of proteins, such as kinases, guanine nucleotide-exchange factors, nucleotide-gated channels, and transcription factors. Moreover, given that proteins containing CNB domains are multimeric, it is also challenging to dissect how the CNB activities are coordinated during cyclic nucleotide signaling.The cyclic-nucleotide binding (CNB) domain is a signaling module found in all kingdoms of life . Its evoFig. 1A). The crystal structures of the R-subunit bound to cAMP (active state) or bound to the PKA catalytic (C) subunit (inactive state) reveal noticeable conformational differences (B). Before reaching the final cAMP-bound conformation, the N3A motif in the cAMP-bound CNB-A domain (denoted as N3AA motif) moves aways from the CNB-A β-subdomain and establishes contacts with the B/C helix and CNB-B domain  subunit of protein kinase A (PKA) contains two CNB domains in tandem (denoted as CNB-A and CNB-B) connected by a helix termed B/C-helix A. The crferences , 3. cAMPferences . Moreoveferences B. BeforeB domain , 5.B), which disrupts interdomain interactions  Disruption of surface contacts between the two CNB domains. (3) Stabilization of the N3AA motif in the distant CNB-A domain. Computational work reveals that the two CNB domains have unique thermodynamic signatures, resulting is different residue connectivity networks despite sharing the same structural makeup. The computational results support the experimental observations of the differential responses of the CNB domains with different binding partners (cAMP or C-subunit). W260A abolishes many of the residue connectivity networks throughout the R-subunit, providing a thermodynamic foundation for the expansive mutational effects of W260A seen experimentally. Altogether, this study underscores the complex interplay between folding energetics, conformational dynamics, and thermodynamic signatures underlying structurally conserved signaling modules in response to ligand binding and mutational effects.Our study shows that the CNB domains exhibit unique folding energy landscape signatures in each of the three functional states, underscoring their plasticity to adapt to different binding partners. The mutation W260A exerts differential effects depending on the functional state. For instance, W260A has no effects on the C-subunit-bound state, moderate effects on the stability of the CNB domains in the apo state, and large effects on the stability and dynamics of the CNB domains in the cAMP-bound state. We find that these mutational effects in the cAMP-bound R-subunit are both local and allosteric: (1) Intra-domain interactions between the N3Avia disulfide bond linkages at positions flanking the two CNB domains (Y120C and S376C) . These molecular trajectories revealed two major sudden changes in extension, or rips, that were assigned to unfolding of each CNB domain of the R-subunit , for the CNB-B domain was slightly lower than the CNB-A for both the wildtype and the mutant R-subunits . To determine which kinetic parameter accounts for the difference in Fave, we extracted the folded state lifetime at zero force (τ0) and the distance to the transition state (Δx‡) of both CNB domains using the Bell model . Due to interdomain interactions triggered by cAMP binding , the wildtype CNB-B domain unfolded at a force higher than that of the truncation, 14.8 ± 1.5 pN and 12.6 ± 0.9 pN, respectively . This observation indicates that the presence of cAMP reshapes the folding energy landscape of the CNB-B domain, energetically decoupling the N3AB motif and the β-subdomain to unfold in two steps instead of a single cooperative one. Interestingly, while the Fave of the CNB domains bound to cAMP was higher than in the apo state, there were no differences between WT and the W260A mutant . Therefore, the simplest interpretation of our results is that the response of the CNB-B domain to cAMP binding is similar to that of the CNB-A domain, where the N3AB motif and the β-subdomain are energetically uncoupled and therefore unfold independent of each other.Previous structural studies on the CNB-A domain showed that the relative orientation of the N3Aubdomain . Our pre, whereas the WT domain exclusively unfolded in a single step . Altogether, we show that the W260A mutation has a pronounced effect on the R-subunit residue networks by destabilizing both the interdomain interactions between the two CNB domains and intradomain interactions between the N3AB motif and the β-subdomain.Next, we investigated how the presence of the CNB-A domain allosterically affects the folding energy landscape of the CNB-B domain bound to cAMP . WLC ana A–C and . In contcontacts E. AltogeA motif unfolds first, followed by the CNB-B domain (N3AB motif and its β-subdomain) and ending with the β-subdomain of CNB-A  .Given the widespread mutational effects of the W260A on the cAMP-bound R-subunit, we further investigated if the mutation exerts allosteric effects over the CNB-A domain itself. In previous mechanical unfolding studies, we have observed that the WT R-subunit unfolds in three transitions: the N3Aof CNB-A A, left  . In the bdomain. A, right.ave for the N3AB motif was the same as those observed for its truncation, 12.79 ± 0.74 pN and 11.29 ± 0.83 pN, respectively . However, the N3AA motif in the mutant R-subunit unfolded ∼2 pN higher than the WT at 14.5 ± 0.9 pN . Given that the two CNB domains in the W260A R-subunit do not establish inter-domain contacts , the most parsimonious interpretation for the observed stabilization of the N3AA motif is long-range allosteric effects. To gain additional insight on long-range mutational effects, we used the software AlloSigMA . However, the calculations revealed major mutational perturbations throughout the R-subunit in the cAMP-bound state. First, the α-helix that harbors W260A mutation was substantially destabilized , in agreement with the experimental results showing the N3AB motif unfolding independently of its β-subdomain. Second, the entire CNB-A domain was stabilized by the mutation with the N3AA motif displaying the strongest effect , which is consistent with our results where the N3AA unfolds at higher forces than in WT. Altogether, the experimental and computational results revealed that, despite structural conservation, the N3A motifs experienced opposite effects due to the W260A mutation.As expected for two non-interacting domains, like the CNB domains in the W260A R-subunit, the Fectively B, left. ± 0.9 pN B, right.lloSigMA C, left. abilized C, in redB and C and Because of the large-scale effects that the W260A mutation exerts over local or distant regions of the R-subunit, we investigated the functional consequences of the mutation in terms of cAMP binding affinities and cooperativities. We performed mechanical unfolding experiments with the mutant R-subunit under different cAMP concentrations to populate four distinct states: apo, intermediate states wherein only the CNB-A or the CNB-B domain is bound to cAMP , and the fully-bound state (termed AB-bound) states . Due to B motif and β-subdomain) unfolds first followed by the CNB-A β-subdomain (A). The partial interdomain interactions triggered by cAMP binding to the CNB-A domain are insufficient to drive folding of the N3AA motif . However, the W260A N3AB motif remained decoupled from its β-subdomain, in agreement with what we observed from the unfolding of the AB-bound state  and the truncated cAMP-bound CNB-B domain (D).In the B-bound state of WT, the N3A motifs and β-subdomains of both CNB domains unfold as single cooperative units, wherein the unbound CNB-A domain unfolds first B. Howevend state A, right B domain D.C, Having characterized the unfolding trajectories of each cAMP-bound species at different cAMP concentrations, we built a single-molecule titration curve and globally fitted the population of each cAMP-bound species to determine cAMP binding affinity constants and cooperativity Fig. 5C,  10). Fo. FoC, 10ave than the apo state but lower than the AB-bound state , the results obtained with optical tweezers show different conformational responses toward cAMP binding. Furthermore, we show that the W260A mutation produces both local and long-range allosteric effects across the CNB domains, and these effects are unique for each domain. These observations motivated us to ask whether each CNB domain in the PKA regulatory subunit has a unique network of thermodynamically coupled residues. Thus, even if the CNB domain structures look alike, their thermodynamic properties and response to mutations may be different. We used COREX/BEST, which is an algorithm that allowed us to calculate thermodynamic coupling as correlated thermodynamic fluctuations between each residue pair in computer-generated protein ensembles  (11). Thi.e., cAMP-bound) display different patterns that map to different parts of their structure . However, for the CNB-A domain, high thermodynamic coupling was largely localized to residues that conform the B/C helix . Importantly, differences in thermodynamic coupling between the CNB domains were also observed in the C-bound state  and as part of the R-subunit . The patterns for thermodynamic coupling of each CNB domain either as part of the R-subunit or as a truncated domain was overall similar, but in certain regions, there were noticeable differences  not only in PKA but in other proteins harboring CNB domains in their structure . The high thermodynamic coupling exclusively localized to the B/C-helix indicates that the core structures of the CNB domains  are largely uncoupled and independent from each other, in agreement with our experimental observations and results using AlloSigMA that show absence of long-range effects due to W260A in the C-bound state . However, an additional region between residues 255 to 268, belonging to the N3AB motif, displayed high thermodynamic coupling in the R-subunit. The high coupling shared between the N3AB motif, and the B/C-helix in the R-subunit indicates the formation of a new cAMP-dependent residue network that not only stabilizes inter-domain contacts with the CNB-A domain but also intra-domain contacts within the CNB-B domain itself. This interpretation agrees with our experimental results in which the N3AB motif unfolds independent of the β-subdomain in the truncated CNB-B domain , whereas in the R-subunit the same domain always unfolds as a single cooperative unit and at a higher force .In the cAMP-bound state, we also observed high thermodynamic coupling around the B/C-helix when the two CNB domains are together B. HoweveB domain E, top, wer force D, top.B motif in the cAMP-bound state displayed high thermodynamic coupling, it is likely that a mutation in this region, such as W260A, will have global effects over the R-subunit. Testing this hypothesis requires an experimentally determined high-resolution structure of the W260A R-subunit, which is not available. However, others have generated this mutant structure using molecular dynamics (MD) simulations . The most significant effects were observed as loss of thermodynamic coupling in the N3AB motif and the B/C helix, in agreement with our experiments showing that the CNB domains in the mutant R-subunit behave as independent domains . In addition, loss of coupling due to W260A was observed in the β-subdomains of both CNB domains.After accounting for the background thermodynamic coupling due to the MD simulation , the heaFig. 7Interestingly, the large-scale mutant effects in the thermodynamic coupling that we observed emerged from differences in the relative orientation of the CNB domains and not because of differences in secondary structures between WT and W260A. For instance, aligning the CNB-A domain (residues 113–242) between the WT and the mutant resulted in a RMSD of 1.01 Å, likewise the alignment for the CNB-B domain (residues 243–376) resulted in 0.97 Å. However, a global alignment with both CNB domains had a RMSD = 5.7 Å. The disparity in RMSD is due to an 18.4-degree difference in rotation of the B/C helix at L233 in the mutant structure, which results in a 6.8 Å overall displacement for the CNB-B domain . Altogeti.e., ligand binding or post-translational modifications), transduce a regulatory signal to other functional domains, and bind multiple protein partners . When bound to cAMP, W260 moves about 30 Å to stabilize the adenine ring of cAMP via π-π stacking. Such interaction is important for communication between the two CNB domains, which explains the significant mutational effect on the R-subunit when bound to cAMP.We find that W260A exerted differential effects on the folding energetics of the CNB domains depending on the functional state. Previous bulk studies reported that W260A disrupts interdomain interactions between the CNB domains , 20. NotFig. 4A motif as a cAMP-dependent molecular switch , or protein kinase G (PKG) where the CNB domains display coordinated structural changes upon cAMP binding , 22, 23.B motif and the CNB-B β-subdomain, where the mutation renders the N3AB motif unable to lock in the final active conformation as in the WT protein. Moreover, the mutation also exerted long-range effects over the N3AA motif, which unfolds at a higher force compared to wildtype (A). These long-range effects were captured by the computational analysis using AlloSigMA, which showed that the stabilized region within the R-subunit due to cAMP binding is highly concentrated to the N3AA motif. All these results indicate that a single mutation can have an expansive effect across the protein, even on regions that are non-interacting.A strength of using optical tweezers to investigate a mutational effect is that we can obtain information on local and long-range effects between non-contiguous protein domains or regions. This technique allows us to dissect the allosteric effects of a single mutation across the PKA R-subunit. Based on the structure, the mutation W260A directly affects interactions that are critical for cAMP binding to the CNB-A domain, which facilitates the conformational reorientation of the CNB domains. Therefore, we expected perturbations would arise from the location of the mutation, namely, loss of interdomain interactions. Less expected was observing the decoupling between the N3Awildtype A. These Thus far, our experimental observations of the CNB domains in the W260A R-subunit indicate that they behave as two non-interacting domains tethered together, which is a behavior different from that observed in the WT protein. We therefore investigated if such a lack of inter-domain interactions would lead to no cooperativity during cAMP binding. We globally analyzed the cAMP titration data and determined a model with no cooperativity was sufficient to fit the data W260A R-subunit. Surprisingly, the binding affinities for cAMP of the mutant protein, both the truncated domains and the R subunit, were higher than the WT truncated CNB domains . This reA and B). In the C-subunit-bound conformation, several residues identified as highly coupled in the B/C-helix  interact with the C-subunit via hydrogen bonds and van der Waals interactions . These results emphasize the role that the B/C-helix plays in interdomain communication beyond residues at the interface of the two CNB domains.Using the WT and W260A mutant R-subunit structures from MD simulations, we determined that the W260A mutation caused an 18.4-degree rotational difference starting at L233 of the B/C-helix. This rotational difference of the B/C-helix may be key to the destabilization between the CNB domains. The increase in flexibility correlates well with the increase in conformational dynamics observed by Guo and Zhou in molecular dynamic simulations  and the et al.  using NMet al. C. These B motif becomes thermodynamically decoupled from the β-subdomain, thereby oscillating between two conformations (①). When the second cAMP binds to the CNB-A domain, the N3AB motif stabilizes the cyclic nucleotide and becomes thermodynamically coupled with the CNB-B β-subdomain, behaving as a single cooperative unit (②). The binding of the second cAMP also leads to the destabilization of the N3AA motif, breaking inte-domain interactions with the CNB-A β-subdomain domain and the C-subunit (③). As the C-subunit dissociates, the N3AA motif stabilizes by establishing contacts with the CNB-B domain and the B/C helix (④). The activation cycle of PKA can be repeated via the removal of cAMP by phosphodiesterase  and the rebinding of the C-subunit with ATP and Mg2+ (⑥). We show that W260A affects the activation pathway of PKA in three different steps: W260A shifts the fluctuation of the N3AB motif towards a decoupled conformation (step ①), there are no inter-domain interactions due to the absence of W260 that serves as cAMP capping residue for the CNB-A domain (step ②), and, W260A stabilizes the N3AA motif allosterically, possibly by retaining intra-domain interactions with the CNB-A β-subdomain (steps ③ and ④). These deficiencies in the conformation of the R-subunit due to W260A prevents the protein from achieving the final, cAMP-bound conformation observed for the WT protein.We provide a comprehensive computational and experimental study on the mutational effect of W260A to the PKA R-subunit in apo, C-subunit-bound, and cAMP-bound states. The combination of experimental and computational analysis reveals that structurally conserved signaling domains have subtle thermodynamic differences and widespread effects due to a mutation. These unique behaviors may emerge from the fact that the CNB domains have evolved to behave as molecular switches, changing in conformation to adapt to the environment, be it a mutation or interacting with a binding partner. Going beyond the CNB domains in the PKA regulatory subunit, the single molecule optical tweezers approach in conjunction with the computational description of protein fluctuations presented in this study can be extended to understand the allosteric effects of mutations in other CNB domain-containing proteins and multi-domain assemblies , 26, 27.via site-directed mutagenesis. The same plasmid was also used to create the truncated CNB-B domain (residue 243–379) and its mutated counterpart. Cysteine residues were modified on residues Y120C/S376C or M243C/S376C on the R-subunit and the truncated CNB-B domain, respectively, for the covalent attachment of double stranded (ds) DNA that serve as molecular handles. The protocols for the purification of PKA R1α, the truncated CNB domains, and the PKA catalytic subunit were previously published  was used for experiments with PKA C-subunit at 100 nM. The main difference between the two buffers was the presence of MgCl2, ATP, and C-subunit, which were critical for the formation of the inactive complex (C-subunit-bound state). The incubation time for protein samples with 3.1 μm anti-digoxigenin coated beads is 15 min with and without cAMP, and 30 min with the C-subunit. The sample mixture is diluted to 1 ml with the same buffer before applying to the microfluidic chamber. The concentration of the DNA–protein chimera in the microfluid chamber is calculated to be below the picomolar range.Protocols for force-ramp experiments with cAMP or with C-subunit were performed as previously published , 29. TheB motif unfolding  and the distance to the transition state in the unfolding reaction (Δxǂ)  :(1)p(F)=(τ) at zero force is calculated from the inverse of k0. See The lifetime p is the persistence length of the polypeptide (0.65 nm) (C) by subtracting the FD between the attachment points in the folded protein from LC.The WLC model was used to determine the number of residues involved in each unfolding event:0.65 nm) , Δx is t0.65 nm) . We repohttp://allosigma.bii.a-star.edu.sg/). The methodology was described by Guarnera et al.  was used in this study. SBSMMA evaluates the allosteric communication upon perturbations such as ligand binding and/or mutations. Here, allosteric effects of the PKA R-subunit bound to the C-subunit or to cAMP due to the W260A mutation were quantified using the AlloSigMA webserver , as well as truncation CNB-B domain (extracted from each PDB) were used to define a harmonic model of the unperturbed  and perturbed  states of the protein domain. The energy function for the unperturbed state is given by0 are the coordinates of the protein structure in generic and reference configurations, respectively; ki,j is the spring constants of the protein harmonic model, and di,j and di,j0 are inter-residue distances. The mutated protein is defined asThe crystal structures of the R subunit bound to the C-subunit or cAMP  and the contact-eliminating mutation (termed down mutation). The difference between the up and down mutations gives rise to the overall allosteric effect.2QCS and 1RGS, respectively), and molecular dynamic simulation structures of WT and W260A R-subunit bound to cAMP were generously provided by Prof. Huan-Xiang Zhou, University of Illinois Chicago . COREX generates a native-state ensemble from the target protein structure through the combined unfolding of adjacent groups of residues defined as folding units, which are treated as native or as unfolded peptides. The free energy of each conformational state within the ensemble is calculated with a surface-area parametrization that has been validated experimentally , 34. Theeference  for detaj,k). ΔΔGj,k represents the long-range effect of the paired residues over each other. The result is visualized in a color-coded N x N matrix, where N is the residue number of the protein. See For each structure , we extracted the associated change in free energy of paired residues (ΔΔGAll data needed to evaluate the conclusions in the paper are present in the paper and/or the This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Radon (Rn) and its decay products are the primary sources of natural ionizing radiation exposure for the public, posing significant health risks, including being a leading cause of lung cancer. Porous material-based adsorbents offer a feasible and efficient solution for controlling Rn concentrations in various scenes to achieve safe levels. However, due to competitive adsorption between Rn and water, finding candidates with a higher affinity and capacity for capturing Rn in humid air remains a significant challenge. Here, we conducted high-throughput computational screening of 8641 two-dimensional covalent organic frameworks (2D COFs) in moist air using grand canonical Monte Carlo simulations. We identified the top five candidates and revealed the structure–performance relationship. Our findings suggest that a well-defined cavity with an approximate spherical inner space, with a diameter matching that of Rn, is the structural basis for a proper Rn capturing site. This is because the excellent steric match between the cavity and Rn maximizes their van der Waals dispersion interactions. Additionally, the significant polarization electrostatic potential surface of the cavity can regulate the adsorption energy of water and ultimately impact Rn selectivity. Our study offers a potential route for Rn management using 2D COFs in moist air and provides a scientific basis for further experimentation. The res−11 ppb . As a re−11 ppb ,5,6. To −11 ppb , pluggin−11 ppb , and ads−11 ppb ,10. AmonCoconut-activated charcoal (AC) is currently the most effective commercialized solid Rn adsorbent, but it also has significant drawbacks. Its ability to maintain a stable and high Rn adsorption level under conditions of high humidity is limited, restricting its widespread use in Rn-rich scenarios with high air humidity, such as water treatment plants, basements, and underground mines. Moreover, due to the lower distribution of Rn size-matching cavities, AC only exhibits moderate Rn capture efficiency and cannot remove Rn thoroughly ,12. InteIn recent years, covalent organic frameworks (COFs) have seen immense popularity as typical porous materials, owing to their unique properties and rapid development ,18,19. TIn this study, we investigated the performance of 8641 2D-layered COFs in the selective adsorption of radon through GCMC simulations. Our results allow us to identified five top-performing candidates with stable and balanced performance in terms of Rn selectivity and adsorption capacity in moist air. Additionally, we reveal the structure–performance relationship of 2D COFs and, importantly, uncover the molecular mechanism by which humidity affects Rn capture performance. These findings not only present a new avenue for Rn capture using 2D COFs but also provide a scientific basis for designing efficient radon trapping materials under high-humidity conditions.Mercado et al.  compiled2/O2/H2O with molar ratios of 0.001/0.776/0.209/0.014, at 298 K and 1 bar. Additionally, we investigated Rn adsorption under various total pressures (10–140 kPa) and humidities , with different Rn concentrations . It is worth noting that the concentration of Rn in the air can differ significantly depending on the location, with values ranging from ~10−11 ppb in common places to ~10−8 ppb in caves or poorly ventilated basements and houses [2, O2, and H2O. Consequently, higher Rn concentrations were conventionally utilized in the GCMC simulations to ensure the comparability of Rn uptake performance among different materials.To evaluate the Rn capture potential of COFs, we conducted GCMC simulations using a moist air mixture of Rn/Nd houses ,31,32. N2 and O2 adsorbates were modeled with the three-site molecular model [2, H2O, and O2) are listed in The GCMC simulations were performed using the RASPA software (version 2.0), as described in a previous study . The Lenar model ,38,39. Tar model . The Rn ar model , based oar model . The fori with respect to gas j, k and m is defined asx and y are the molar ratios of adsorbed species in the adsorbed and bulk phases, respectively [The adsorption of radon obtained from GCMC simulation is the absolute adsorption amount (ectively . In addiectively  was intr2/O2/H2O) to mimic radon exposure in moist air. To assess the performance of the adsorbent materials, we adopted two indexes, Rn selectivity and Rn uptake capacity, as they are crucial for conducting cost-effective gas separation processes [Moisture is a critical factor that affects the adsorption of noble gas by porous materials . Thus, irocesses .2 and O2, respectively. It is evident from RnAPS, respectively. Based on the adsorption performance results, we screened the top five COFs with the highest RnAPS values (>190 mol/kg), including linker105_C_linker13_C_kgm (~300.85 mol/kg) > linker101_C_linker11_C_kgm (~287.26 mol/kg) > linker107_C_linker13_C_kgm (~215.24 mol/kg) linker99_C_linker11_C_kgm (~198.26 mol/kg) > linker99_C_linker13_C_kgm (~193.60 mol/kg). The IUPAC names and structures of the linker groups in the top five COFs can be found in 3 to 123.56 Å3, demonstrating an excellent stereo shape matching the spherically shaped Rn (4.17 Å). This observation is consistent with the principle that cavities with geometries matching those of Rn are more thermodynamically favorable for accommodating Rn atoms, due to the relatively strong vdW dispersion interactions. A similar phenomenon was also observed in the case of ZIF-7, which has a Rn-matching spherical cage with a diameter of ~4.38 Å and displays remarkable Rn selectivity and uptake capacity.In order to establish a correlation between the structural features of COFs and their performance in capturing Rn, we conducted a detailed analysis of the spatial distributions of Rn in the top five candidates. This allowed us to identify the most preferential binding sites for Rn. Interestingly, our findings, as illustrated in 2/O2 was maintained at 0.001/0.776/0.209, while the RH values were varied from 0 to 1.0.As previously discussed, moisture can significantly impact the Rn capture performance of 2D COFs  . These cAt RH = 0.0, both candidates exhibited two major Rn binding sites  . RemarkaThe structural features of binding site i in linker105_C_linker13_C_kgm enable it to provide greater energy gains from electrostatic interactions with water. As depicted in −11 ppb) is often beyond the numerical limit of most GCMC software (single-precision floating-point format). Hence, a relatively high gradient of molar concentration was employed to investigate the impact of gas concentration variations on nanoporous material performance [RnX , where moisture represents 0.5 (waterX = 0.014) at ambient temperature, and the molar ratio of formance ,14. TherRnX was below 0.0005, the Rn uptake capacity of linker105_C_linker13_C_kgm, linker107_C_linker13_C_kgm, and linker99_C_linker13_C_kgm was almost at the same level, while linker101_C_linker11_C_kgm and linker99_C_linker11_C_kgm had a comparable Rn capacity, albeit slightly lower than the former three candidates. At the highest Rn molar fraction (RnX = 0.001), the differences in the performances of the COFs became apparent, with the Rn uptake capacity order being linker105_C_linker13_C_kgm > linker107_C_linker13_C_kgm > linker99_C_linker13_C_kgm = linker99_C_linker11_C_kgm > linker101_C_linker11_C_kgm.As shown in RnX = 0.001, with pressures ranging from 5 to 140 kPa. As shown in We also evaluated the Rn-capture isothermal adsorption curves of screened five COFs under RH = 0.5 at room temperature. 2/O2/H2O of 0.001/0.776/0.209/0.014 at ambient temperature and pressure. After thorough evaluation, we identified five COFs, namely linker105_C_linker13_C_kgm, linker101_C_linker11_C_kgm, linker107_C_linker13_C_kgm, linker99_C_linker11_C_kgm, and linker99_C_linker13_C_kgm, as the top candidates for Rn capture. The analysis of structure–performance relationships suggested that the well-defined cavities in COFs, having a diameter that matches that of Rn, represent a very crucial prerequisite for competitive Rn capturing sites because the excellent steric match between the cavity and Rn could maximize their vdW dispersion interactions for the adsorption energy.In this study, we investigated the potential of 8641 two-dimensional covalent organic frameworks (COFs) to capture radon (Rn) under humid air conditions. To achieve this, high-throughput GCMC simulations were used to screen the COFs based on their Rn capture capability from a quaternary mixed gas with a molar ratio of Rn/NRnX = 0.001), the five screened COFs exhibited slightly lower Rn capacity (ranging from ~0.73 to ~0.96 mol/kg) but much better Rn selectivity (ranging from ~244.1 to ~392.5) compared to ZIF7-Im-1 . To the best of our knowledge, experimentally, ZIF7-Im-1 may remain the record holder for both the highest Rn uptake capacity and selectivity so far [We further evaluated the Rn capture performance of the selected COFs under different humidity conditions . The results showed that both the Rn selectivity and adsorption capacity of all five COFs decreased with increasing RH, with a more pronounced effect observed at lower humidity levels (RH < 0.3). Interestingly, the impact of humidity on the performance of COFs exhibited individual differences. For instance, RH had a greater influence on the performance of linker105_C_linker13_C_kgm than it did on the performance of linker101_C_linker11_C_kgm. The underlying molecular mechanism is attributed to the architecture of polar groups in the binding sites. For instance, in binding site i of linker101_C_linker11_C_kgm, there are six protruding hydroxyl groups that collectively point to the interior space of the cavity, inducing a notable-polarization ESP surface that offers more favorable electrostatic interactions for water. However, in the other three binding sites, the spatial distributions of the polar groups point outward from the cavity  or are arranged in a planar-like manner , which cannot induce the remarkable-polarization ESP surfaces of the cavities and are hence less favorable for electrostatic interactions with water. This is the main molecular mechanism by which RH impacts the Rn capturing performance of COFs. Lastly, we evaluated the influence of Rn molar fractions and pressures on the performance of the COFs. It should be noted that, under equal conditions (at RH = 0.5, 1 bar, 298 K, and y so far . Given t