Site Ligands Research Articles

Using Protein Painting Mass Spectrometry to Define Ligand Receptor Interaction Sites for Acetylcholine Binding Protein.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels expressed in nervous and non-nervous system tissue important for memory, movement, and sensory processes. The pharmacological targeting of nAChRs, using small molecules or peptides, is a promising approach for the development of compounds for the treatment of various human diseases including inflammatory and neurogenerative disorders such as Alzheimer's disease. Using the Aplysia californica acetylcholine binding protein (Ac-AChBP) as an established structural surrogate for human homopentameric α7 nAChRs, we describe an innovative protein painting mass spectrometry (MS) method that can be used to identify interaction sites for various ligands at the extracellular nAChR site. We describe how the use of small molecule dyes can be optimized to uncover contact sites for ligand-protein interactions based on MS detection. Protein painting MS has been recently shown to be an effective tool for the identification of residues within Ac-AChBP involved in the binding of know ligands such as α-bungarotoxin. This strategy can be used with computational structural modeling to identify binding regions involved in drug targeting at the nAChR. Key features • Identify binding ligands of nicotinic receptors based on similarity with the acetylcholine binding protein. • Can be adapted to test various ligands and binding conditions. • Mass spectrometry identification of specific amino acid residues that contribute to protein binding. • Can be effectively coupled to structural modeling analysis.

Construction of an Indium-based Coordination Polymer with Redox Non-Innocent Ligand for High-Efficient Electrochemical CO2 Reduction.

Developing high-activity and long-term stable electrocatalysts for electrochemical CO2 reduction reaction (eCO2RR) to valuable products is still a challenge. An in-depth understanding of reaction mechanisms and the structure-function relationship is required for the development of an advanced catalytic eCO2RR system. Herein, a coordination polymer of indium(III) and benzenehexathiol (BHT) was developed as an electrocatalyst (In-BHT) for eCO2RR to HCOO-, which displayed an outstanding catalytic performance over the entire pH range. However, experimental results revealed significantly different catalytic pathways in the acid and neutral/alkaline solutions, which are attributed to the influence of redox non-innocent ligands on the rate-determining step (RDS). In the acid solution, the RDS is the formation of *OCOH intermediate through the proton transfer that originates from H2O in the solution, leading to relatively sluggish kinetics. But in the neutral or alkaline solution, the thiolate groups could be protonated during the catalytic process, and such proton can attack on carbon of absorbed CO2 via an intramolecular proton transfer, promoting the formation of *OCHO intermediate, resulting in faster kinetics. Our findings revealed the pivotal roles of the redox non-innocent ligands of metal active sites for eCO2RR, providing a new idea for designing highly efficient electrocatalysts.

PPDock: Pocket Prediction-Based Protein-Ligand Blind Docking.

Predicting the docking conformation of a ligand in the protein binding site (pocket), i.e., protein-ligand docking, is crucial for drug discovery. Traditional docking methods have a long inference time and low accuracy in blind docking (when the pocket is unknown). Recently, blind docking techniques based on deep learning have significantly improved inference efficiency and achieved good docking results. However, these methods often use the entire protein for docking, which makes it difficult to identify the correct pocket and results in poor generalization performance. In this study, we propose a two-stage docking paradigm, where pocket prediction is followed by pocket-based docking. Following this paradigm, we design a new blind docking method based on pocket prediction (PPDock). Through extensive experiments on benchmark data sets, our proposed PPDock outperforms existing methods in nearly all evaluation metrics, demonstrating strong docking accuracy, generalization ability, and efficiency.

Conformational Dynamics of Mitochondrial Inorganic Pyrophosphatase hPPA2 and Its Changes Caused by Pathogenic Mutations.

Inorganic pyrophosphatases, or PPases, are ubiquitous enzymes whose activity is necessary for a large number of biosynthetic reactions. The catalytic function of PPases is dependent on certain conformational changes that have been previously characterized based on the comparison of the crystal structures of various complexes. The current work describes the conformational dynamics of a structural model of human mitochondrial pyrophosphatase hPPA2 using molecular dynamics simulation, all-atom principal component analysis, and coarse-grained normal mode analysis. In addition to the wild-type enzyme, four mutant variants of hPPA2 were characterized that correspond to the natural pathogenic variants causing severe mitochondrial dysfunction and cardio pathologies. As a result, we identified the global type of flexible motion that seems to be shared by other dimeric PPases. This motion is discussed in terms of the allosteric behavior of the protein. Analysis of the observed conformational dynamics revealed the formation of a binding site for anionic ligands in the active site that could be relevant to enzyme catalysis. Based on the comparison of the wild-type and mutant PPases dynamics, we suggest the possible molecular mechanisms of the functional incompetence of hPPA2 caused by mutations. The results of this work allow for deeper insight into the structural basis of PPase function and the possible effects of pathogenic mutations on the protein structure and function.

Tipping the balance between twist and chair ligand conformers in multinuclear ethylzinc cyclophosphazenates.

Hexaanionic cyclophosphazenate ligands [(RN)6P3N3]6- provide versatile platforms for the assembly of multinuclear metal arrays due to their multiple coordination sites and highly flexible ligand core structure. This work investigates the impact of incrementally increasing the steric demand of the ligand periphery on the coordination behavior of ethylzinc arrays. It shows that the increased congestion around the ligand sites is alleviated by progressive condensation with the elimination of diethylzinc. The condensation is accompanied by a switch in ligand conformation: the phosphazene ring of the methyl derivative adopts the chair conformer in the dimeric complex (EtZn)12[(RN)6P3N3]2, while the more sterically demanding isobutyl derivative exhibits twist conformers in both the monomeric complex (EtZn)6[(iBuN)6P3N3] and the condensed dimer (EtZn)8Zn2[(iBuN)6P3N3]2. Ethyl and propyl derivatives mark a tipping point: the dimeric complex (EtZn)12[(RN)6P3N3]2 exhibits the chair conformation and is observed in solution and within the stabilising environment of a co-crystal. However, when crystallising in pure form, it transforms with the loss of one equivalent of Et2Zn into the collapsed dimer (EtZn)10Zn[(RN)6P3N3]2 which features the twist conformer.

Mechanically Robust, Superlubricating and Antifouling Bilayer Nanocoating for Micro-Bioimplants via a Dual-Function Metal Coordination Approach.

Nanometer-thick ultrathin coatings with superior mechanical strength and desirable lubricating and antifouling performance are critical for the miniaturization of implantable medical devices. However, integrating these properties at the nanoscale remains challenging due to the inherent trade-off between mechanical strength and hydration as well as limitations in coating thickness. In this work, we address these challenges by employing dual-function metal coordination to construct a ∼25 nm thick bilayer structure. Contact mechanics and interfacial molecular force measurements confirm the dual role of vanadium (VIII) ions in forming this bilayer: VIII ions bridge the ligand sites to reinforce the protein bottom layer, and simultaneously anchor the end blocks of the designed ABA triblock hydrophilic polymers to form a hydrated, looping top layer. This VIII-enabled structure demonstrates remarkable load-bearing capacity and lubricating performance (i.e., friction coefficient μ on the order of 10-3 over 100 cycles under ∼10 MPa), while it also exhibits excellent resistance to biofouling in complex biological fluids. This work presents a useful strategy for integrating seemingly incompatible properties into ultrathin coatings, offering the potential for customizing multifunctional surfaces for micro-devices/machines toward bioengineering applications.

Coexistence of Redox-Active Metal and Ligand Sites in Copper-Based Two-Dimensional Conjugated Metal-Organic Frameworks as Active Materials for Battery-Supercapacitor Hybrid Systems.

Two-dimensional (2D) conjugated metal-organic frameworks (c-MOFs) are promising materials for supercapacitor (SC) electrodes due to their high electrochemically accessible surface area coupled with superior electrical conductivity compared to traditional MOFs. In this work, porous and non-porous HHB-Cu (HHB=hexahydroxybenzene), derived through surfactant-assisted synthesis are studied as representative 2D c-MOF models with different characteristics, showing diverse reversible redox reactions with Na+ and Li+ in aqueous (10 M NaNO3) and organic (1.0 M LiPF6 in ethylene carbonate and dimethyl carbonate) electrolytes, respectively. These redox activities were here deployed to design negative electrodes for hybrid SCs (HSCs), combining the battery-like property of HHB-Cu at the negative electrode and the high capacitance and robust cyclic stability of activated carbon (AC) at the positive electrodes. In the organic electrolyte, porous HHB-Cu-based HSC achieves a maximum cell specific capacity (Cs) of 22.1 mAh g-1 at 0.1 A g-1, specific energy (Es) of 15.55 Wh kg-1 at specific power (Ps) of 70.49 W kg-1, and 77 % cyclic stability after 3000 gravimetric charge-discharge (GCD) cycles at 1 A g-1 (specific metrics calculated on the mass of both electrode materials). In the aqueous electrolyte, porous HHB-Cu-based HSC displays a Cs of 13.9 mAh g-1 at 0.1 A g-1, Es of 6.13 Wh kg-1 at 44.05 W kg-1, and 72.3 % Cs retention after 3000 GCD cycles. The non-porous sample, interesting for its superior electrical conductivity despite its limited surface area compared to its porous counterpart, shows lower Es performance but better rate capability compared to the porous one. This study indicates the potential of assembling a battery-SC hybrid system by rationally exploiting the battery-like behavior of 2D c-MOFs and the electrochemical double-layer capacitance of AC.

Insights and progress on the biosynthesis, metabolism, and physiological functions of gamma-aminobutyric acid (GABA): a review.

GABA (γ-aminobutyric acid) is a non-protein amino acid that occurs naturally in the human brain, animals, plants and microorganisms. It is primarily produced by the irreversible action of glutamic acid decarboxylase (GAD) on the α-decarboxylation of L-glutamic acid. As a major neurotransmitter in the brain, GABA plays a crucial role in behavior, cognition, and the body's stress response. GABA is mainly synthesized through the GABA shunt and the polyamine degradation pathways. It works through three receptors (GABAA, GABAB, and GABAC), each exhibiting different pharmacological and physiological characteristics. GABA has a variety of physiological roles and applications. In plants, it regulates growth, development and stress responses. In mammals, it influences physiological functions such as nervous system regulation, blood pressure equilibrium, liver and kidneys enhancement, hormone secretion regulation, immunity enhancement, cancer prevention, as well as anti-aging effects. As a biologically active ingredient, GABA possesses unique physiological effects and medicinal value, leading to its widespread application and substantially increased market demand in the food and pharmaceutical industries. GABA is primarily produced through chemical synthesis, plant enrichment and microbial fermentation. In this review, we first make an overview of GABA, focusing on its synthesis, metabolism, GABA receptors and physiological functions. Next, we describe the industrial production methods of GABA. Finally, we discuss the development of ligands for the GABA receptor binding site, the prospects of GABA production and application, as well as its clinical trials in potential drugs or compounds targeting GABA for the treatment of epilepsy. The purpose of this review is to attract researchers from various fields to focus on GABA research, promote multidisciplinary communications and collaborations, break down disciplinary barriers, stimulate innovative research ideas and methods, and advance the development and application of GABA in medicine, agriculture, food and other fields.

Engineering Logic‐Gated i‐Motif/G‐Quadruplex (iG4) Hybrid Structures for DNA Nanotweezer‐Based Aptasensing

AbstractGiven high levels of both K+ and H+ in the tumor microenvironment, G‐quadruplex (G4) and i‐motif can in principle be utilized as two cooperative modules to build logic‐gated DNA nanodevices for microenvironment recognition and targeting. Combined use of G4 and i‐motif in DNA nanoassemblies, however, usually causes the uncontrolled DNA aggregation. To address this trouble, we well design a novel i‐motif/G‐quadruplex (iG4) hybrid structure that integrates two four‐stranded DNA helices in a folding topology, with a parallel mini‐duplex flanking at the 5’ end to provide a binding site for fluorescent ligands. This design enables that the folded and lighting‐up DNA structure is favored by H+ and K+ together, consistent with a two‐input AND gate behavior. We then employ the iG4 structure to guide a DNA nanotweezer that is clamped by an altered split ATP aptamer, which brings a proximity effect contributing to the proper folding of iG4 in slightly acidic microenvironments and enables the sensitive detection of endogenous ATP in cancer cell lysates.

Specific radiation damage to halogenated inhibitors and ligands in protein-ligand crystal structures.

Protein-inhibitor crystal structures aid medicinal chemists in efficiently improving the potency and selectivity of small-molecule inhibitors. It is estimated that a quarter of lead molecules in drug discovery projects are halogenated. Protein-inhibitor crystal structures have shed light on the role of halogen atoms in ligand binding. They form halogen bonds with protein atoms and improve shape complementarity of inhibitors with protein binding sites. However, specific radiation damage (SRD) can cause cleavage of carbon-halogen (C-X) bonds during X-ray diffraction data collection. This study shows significant C-X bond cleavage in protein-ligand structures of the therapeutic cancer targets B-cell lymphoma 6 (BCL6) and heat shock protein 72 (HSP72) complexed with halogenated ligands, which is dependent on the type of halogen and chemical structure of the ligand. The study found that metrics used to evaluate the fit of the ligand to the electron density deteriorated with increasing X-ray dose, and that SRD eliminated the anomalous signal from brominated ligands. A point of diminishing returns is identified, where collecting highly redundant data reduces the anomalous signal that may be used to identify binding sites of low-affinity ligands or for experimental phasing. Straightforward steps are proposed to mitigate the effects of C-X bond cleavage on structures of proteins bound to halogenated ligands and to improve the success of anomalous scattering experiments.

Site-differentiated ligand substitutions of the incomplete-cubane type W-Fe-S clusters

A comprehensive study of the reactivity at the core bridging sites and the terminal site of Fe-S cluster will provide a useful reference for understanding the potential N2 binding sites in FeMo-cofactor. While traditional Fe-S clusters exhibit no chemical reactivity within the core due to the saturated Fe coordination by S-containing ligands, the previously synthesized incomplete-cubane cluster [(Tp*)WFe2S3(η-Cl)2(μ2-Cl)]− (1) [Tp*=tris(3,5-dimethyl-1-pyrazolyl)hydroborate(1−)] provides a good platform for such investigations. In this work, the reactivity of the cluster at different ligand sites within and outside the core has been studied. The µ2-bridging chloride within the core demonstrates higher reactivity toward ligand metathesis by methanethiolate or thiophenolate ligand, as compared with the terminally bound chlorides outside the core. Interestingly, the reactivity of the bridging and terminal chlorides could not be differentiated when p-thiocresolate is employed as the substituent for room temperature reaction. Single-crystal structures and redox behaviors of the thus synthesized W-Fe-S clusters have been studied systematically.

Self‐Assembled Antibody‐Oligonucleotide Conjugates for Targeted Delivery of Complementary Antisense Oligonucleotides

AbstractAntibody‐oligonucleotide conjugate (AOC) affords preferential cell targeting and enhanced cellular uptake of antisense oligonucleotide (ASO). Here, we have developed a modular AOC (MAOC) approach based on accurate self‐assembly of separately prepared antibody and ASO modules. Homogeneous multimeric AOC with defined ASO‐to‐antibody ratio were generated by L–DNA scaffold mediated precise self‐assembly of antibodies and ASOs. The MAOC approach has been implemented to deliver exon skipping ASOs via transferrin receptor (TfR1) mediated internalization. We discovered an anti‐TfR1 sdAb that can greatly enhance nuclear delivery of ASOs. Cryo‐EM structure of the sdAb‐TfR1 complex showed a new epitope that does not overlap with the binding sites of endogenous TfR1 ligands. In vivo functional analyses of MAOCs with one ASO for single exon skipping and two ASOs for double exon skipping showed that both ASO concentration and exon skipping efficacy of MAOC in cardiac and skeletal muscles are dramatically higher than conventional ASOs in the transgenic human TfR1 mouse model. MAOC treatment was well tolerated in vivo and not associated with any toxicity‐related morbidity or mortality. Collectively, our data suggest that the self‐assembled MAOC is a viable option for broadening the therapeutic application of ASO via multi‐specific targeting and delivery.

Lysosomal enzyme binding to the cation-independent mannose 6-phosphate receptor is regulated allosterically by insulin-like growth factor 2.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor's conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

Self-Assembled Antibody-Oligonucleotide Conjugates for Targeted Delivery of Complementary Antisense Oligonucleotides.

Antibody-oligonucleotide conjugate (AOC) affords preferential cell targeting and enhanced cellular uptake of antisense oligonucleotide (ASO). Here, we have developed a modular AOC (MAOC) approach based on accurate self-assembly of separately prepared antibody and ASO modules. Homogeneous multimeric AOC with defined ASO-to-antibody ratio were generated by L-DNA scaffold mediated precise self-assembly of antibodies and ASOs. The MAOC approach has been implemented to deliver exon skipping ASOs via transferrin receptor (TfR1) mediated internalization. We discovered an anti-TfR1 sdAb that can greatly enhance nuclear delivery of ASOs. Cryo-EM structure of the sdAb-TfR1 complex showed a new epitope that does not overlap with the binding sites of endogenous TfR1 ligands. In vivo functional analyses of MAOCs with one ASO for single exon skipping and two ASOs for double exon skipping showed that both ASO concentration and exon skipping efficacy of MAOC in cardiac and skeletal muscles are dramatically higher than conventional ASOs in the transgenic human TfR1 mouse model. MAOC treatment was well tolerated in vivo and not associated with any toxicity-related morbidity or mortality. Collectively, our data suggest that the self-assembled MAOC is a viable option for broadening the therapeutic application of ASO via multi-specific targeting and delivery.

Dynamic Coordination Engineering of Z‐Scheme (FFV)2PdCl2/C3N4 Heterojunction for Superior Photocatalytic Hydrogen Evolution

AbstractRealizing highly efficient photocatalytic hydrogen evolution reaction (HER) is a key challenge. Herein, a (FFV)2PdCl2 complex is developed with dynamic coordination engineering between the PdII site and Fluoflavin (FFV) ligands, and couple it with graphite carbon nitride (g‐C3N4) ultrathin nanosheets to construct a novel Z‐scheme heterojunction ((FFV)2PdCl2/C3N4). The resultant heterojunction delivers a HER activity of 648 µmol h−1 under visible light (λ ≥ 400 nm) illumination and an apparent quantum yield up to 40.1% at 400 nm, far superior to those g‐C3N4‐based catalysts reported previously. Mechanistic and theoretical studies reveal that the dynamic coordination between the PdII site and FFV ligands not only significantly accelerates the electron transfer from g‐C3N4 to (FFV)2PdCl2 and then to the PdII sites via a Z‐scheme mechanism, but also effectively maintain the efficacy and stability of the PdII active sties, and thus the (FFV)2PdCl2/C3N4 with a ultralow Pd‐loading amount (ca. 0.1 wt.%) exhibits the impressive activity and durability. The present dynamic coordination and structural evolution of (FFV)2PdCl2 are also applicable for significantly improving the HER performance of other semiconductors, thus paving a potential way for manufacturing highly efficient and active H2 production systems.

Identification and Ranking of Binding Sites from Structural Ensembles: Application to SARS-CoV-2.

Target identification and evaluation is a critical step in the drug discovery process. Although time-intensive and complex, the challenge becomes even more acute in the realm of infectious disease, where the rapid emergence of new viruses, the swift mutation of existing targets, and partial effectiveness of approved antivirals can lead to outbreaks of significant public health concern. The COVID-19 pandemic, caused by the SARS-CoV-2 virus, serves as a prime example of this, where despite the allocation of substantial resources, Paxlovid is currently the only effective treatment. In that case, significant effort pre-pandemic had been expended to evaluate the biological target for the closely related SARS-CoV. In this work, we utilize the computational hot spot mapping method, FTMove, to rapidly identify and rank binding sites for a set of nine SARS-CoV-2 drug/potential drug targets. FTMove takes into account protein flexibility by mapping binding site hot spots across an ensemble of structures for a given target. To assess the applicability of the FTMove approach to a wide range of drug targets for viral pathogens, we also carry out a comprehensive review of the known SARS-CoV-2 ligandable sites. The approach is able to identify the vast majority of all known sites and a few additional sites, which may in fact be yet to be discovered as ligandable. Furthermore, a UMAP analysis of the FTMove features for each identified binding site is largely able to separate predicted sites with experimentally known binders from those without known binders. These results demonstrate the utility of FTMove to rapidly identify actionable sites across a range of targets for a given indication. As such, the approach is expected to be particularly useful for assessing target binding sites for any emerging pathogen, as well as for indications in other disease areas, and providing actionable starting points for structure-based drug design efforts.

Synthesis, Crystal Structure, DFT Calculations, Bioactivity Study, and Docking Results of Bis‐Hydrazone Based on 1,2,4‐Triazol‐3‐Thione

AbstractThe new, unexpected bioactive bis‐hydrazone derivative (4) was obtained, in 74 % yield, by reacting two molar equivalents of pyrazole‐4‐carbaldehyde (1) with one molar equivalent of 2,5‐dihydrazineyl‐1,3,4‐thiadiazole (2). The compound was comprehensively characterized, including X‐ray single crystal, DFT calculations, and bioactivity assessments. Hirschfeld surface analysis confirmed the presence of hydrogen bonding interactions, particularly N−H⋅⋅⋅N and C−H⋅⋅⋅π interactions, which influence the overall crystal packing. The target bis‐hydrazone exhibited significant antimicrobial activity against multi‐drug‐resistant bacterial strains, with the largest activity against S.typhimurium with an inhibition zone of 17.1±0.6 mm and MIC 31.25 μg/mL. The compound also demonstrated significant cytotoxic effects on cancer cells, with a higher IC50 ratio of 134.43 μg/mL against the normal cell line Wi38 and the lowest IC50 value of 45.88 μg/mL against the cancer cell line Caco2. Molecular docking was carried out with estrogen receptor alpha (ERα) and sodium‐glucose transporter SGLT1, which are relevant to Mcf7 and Caco2 cancer cell lines, respectively. Docking suggests the presence of specific amino acids that may influence the binding affinity between the ligand and receptor active sites through residue overlaps in chains A for SGLT1 and B for Erα, offering the ligand as a promising anticancer consistent with the IC50 outcomes.

Heteroatomic-scale insight into the extraction selectivity of amic acid ligands and gallium and indium recovery from spent solar panels

The rapid growth of traditional electronic and emerging renewable energy devices has compelled an increasing global demand for critical metals, such as gallium (Ga) and indium (In). Extractants capable of capturing Ga and In from complex sources, especially the generated spent devices, are thus needed for the critical metals sustainability and the spent devices recycling. However, developing an environmentally benign extractant with high selectivity remains a challenge. Here, a method for regulating the extraction selectivity of “green” amic acid ligands toward Ga3+ and In3+ by heteroatom (S, O and N) substitution was proposed and verified by experimental and theoretical calculations. The experimental results indicated that heteroatom substitution contributed to a great increase of Ga3+ and In3+ extraction capacities, and the hard O-substituted amic acid ligand (AA-O) exhibited outstanding selectivity in separating Ga3+ and In3+ from the competing metal ions of Al3+, Cu2+, Cu2+ and Zn2+. The theoretical calculations showed that heteroatoms made the dominant contribution to the extraction selectivity over amide and carboxyl coordination sites in amic acid ligands. Based on these findings, the AA-O ligand was employed as the optimal extractant to recover Ga3+ and In3+ from the leachate of spent solar cells via a single-step extraction process, without the use of additional extractants. The successful separation of Ga3+ and In3+, with purity improvement from 8.73 and 21.39 wt% to 78.31 and 97.05 wt%, respectively, suggested that AA-O ligand is a promising extractant and supported the feasibility of selectivity regulation for amic acid ligands via heteroatom substitution.

D1-Tyr246 and D2-Tyr244 in photosystem II: Insights into bicarbonate binding and electron transfer from QA•− to QB

In photosystem II (PSII), D1-Tyr246 and D2-Tyr244 are symmetrically located at the binding site of the bicarbonate ligand of the non-heme Fe complex. Here, we investigated the role of the symmetrically arranged tyrosine pair, D1-Tyr246 and D2-Tyr244, in the function of PSII, by generating four chloroplast mutants of PSII from Chlamydomonas reinhardtii: D1-Y246F, D1-Y246T, D2-Y244F, and D2-Y244T. The mutants exhibited altered photoautotrophic growth, reduced PSII protein accumulation, and impaired O2-evolving activity. Flash-induced fluorescence yield decay kinetics indicated a significant slowdown in electron transfer from QA•− to QB in all mutants. Bicarbonate reconstitution resulted in enhanced O2-evolving activity, suggesting destabilization of bicarbonate binding in the mutants. Structural analyses based on a quantum mechanical/molecular mechanical approach identified the existence of a water channel that leads to incorporation of bulk water molecules and destabilization of the bicarbonate binding site. The water intake channels, crucial for bicarbonate stability, exhibited distinct paths in the mutants. These findings shed light on the essential role of the tyrosine pair in maintaining bicarbonate stability and facilitating efficient electron transfer in native PSII.

Macroporous cellulose microspheres with high specific surface area via semi-dissolved chitosan templating for protein separation

The conflict between macroporous structure and high specific surface area strongly limited the dynamic adsorption performance of chromatographic media. Here, macroporous and high-specific-surface-area cellulose microspheres (PCMs) are prepared by using semi-dissolved chitosan as a dual-functional template and subsequently functionalized with diethylaminoethyl (DEAE) as anion exchangers for protein separation. The dual-functional template strategy leverages dissolved chitosan nanofibers as meso/micropore templates and the undissolved chitosan particles as macropore templates. The macropores of DEAE-PCMs (average size: 0.4–1.2 μm) efficiently decrease mass transfer resistance for fast uptake kinetics. The high specific surface area (272.34 m2/g) provides sufficient ligand sites for ensuring exceptional adsorption capacities. Therefore, the chitosan bifunctional templated PCMs, possessing a macro/meso/microporous structure, are promising candidates for high-performance chromatographic media.