ACS Publications最新文献

{"title":"Chipless Millifluidics Device for Rapid Fabrication of Hepatic Spheroids with Decellularized Liver Matrix.","authors":"Sourita Ghosh, Amit Ghosh, Falguni Pati, Suhanya Duraiswamy","doi":"10.1021/acsbiomaterials.5c01058","DOIUrl":"https://doi.org/10.1021/acsbiomaterials.5c01058","url":null,"abstract":"<p><p>Spheroids - simple 3D cellular aggregates, provide a superior model for simulating the <i>in vivo</i> complexity and architecture often lacking in traditional 2D cell cultures. They have numerous applications, including drug toxicity testing, disease modeling, tumor research, and regenerative medicine. However, existing spheroid preparation methods face significant challenges, particularly in achieving consistent shapes and sizes. Our work focuses on developing an affordable, user-friendly, and robust device for automated spheroid generation. This device uniquely utilizes decellularized extracellular liver matrix to encapsulate HepG2 cells, producing uniform liver spheroids as precise droplets in virgin coconut oil. The novelty of our approach lies in its ability to quickly and efficiently produce spheroids in large quantities, allowing for reproducible spheroid production while addressing the limitations of standard methods. By enhancing control over spheroid morphology and facilitating large-scale generation, our device promises to significantly advance the field of <i>in vitro</i> modeling and improve the reliability of experimental outcomes in various biomedical applications.</p>","PeriodicalId":8,"journal":{"name":"ACS Biomaterials Science & Engineering","volume":" ","pages":""},"PeriodicalIF":5.5,"publicationDate":"2025-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterizing Infrared Spectra of OH^-·(H₂O)₂and OH^-·(H₂O)₃ with Constrained Nuclear-Electronic Orbital Molecular Dynamics.","authors":"Zhe Liu, Yiwen Wang, Yuzhe Zhang, Nan Yang, Yang Yang","doi":"10.1021/acs.jpca.5c04334","DOIUrl":"https://doi.org/10.1021/acs.jpca.5c04334","url":null,"abstract":"<p><p>The vibrational spectra of OH^-·(H₂O)_<i>n</i> clusters for small <i>n</i> have been well established experimentally, with fundamental modes largely assigned. However, clear assignment of highly anharmonic modes and combination bands associated with strong hydrogen bonds, which often manifest as broad spectral features, remains challenging. In this work, we employ constrained nuclear-electronic orbital molecular dynamics (CNEO-MD) to provide detailed peak assignments and plausible physical interpretations for the vibrational spectra of OH^-·(H₂O)_n clusters with <i>n</i> = 2 and 3. The CNEO framework incorporates nuclear quantum effects, particularly nuclear quantum delocalization, through the underlying effective potential energy surfaces. When combined with classical molecular dynamics, CNEO-MD further captures coupling effects between vibrational modes. Leveraging machine-learned potentials, we perform a series of temperature-dependent CNEO-MD simulations and use the resulting spectra to facilitate peak assignment. Our results largely confirm the experimental assignments reported by Johnson and coworkers [<i>J. Chem. Phys.</i> <b>2016</b>, 145, 134304], while also providing direct, physically grounded interpretations of previously unassigned features.</p>","PeriodicalId":59,"journal":{"name":"The Journal of Physical Chemistry A","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Altered Abundance of Barrier-Related Proteins in Brain Microvascular Endothelial Cells of the GL261 Mouse Model of Glioblastoma.","authors":"Liam M Koehn, Diana Cao, Joel R Steele, Angela Rigopoulos, Ingrid Jg Burvenich, Han Chung-Lee, Erwin Tanuwidjaya, Ralf B Schittenhelm, Andrew M Scott, Hui K Gan, Joseph A Nicolazzo","doi":"10.1021/acs.molpharmaceut.5c00609","DOIUrl":"https://doi.org/10.1021/acs.molpharmaceut.5c00609","url":null,"abstract":"<p><p>Glioblastoma multiform (GBM) is a brain cancer that has limited treatment options and a high fatality rate, due in part to limited access of chemotherapeutics to the tumor resulting from the restrictive nature of the blood-brain barrier (BBB). The present study characterized the proteome of endothelial cells forming the BBB in a mouse model of GBM, as a way to identify putative transporters that could be exploited to enhance drug delivery in GBM. Female 6-8 week old C57BL/6 mice were intracranially injected with glioma 261 (GL261) cells or underwent a sham injection. After 28-29 days, brain endothelial cells (CD31+/CD45-) from GL261 (GBM-EC) and sham-injected (control-EC) mice were isolated using magnetic-activated cell sorting, and the proteome of cells was compared by untargeted liquid chromatography dual mass spectrometry. GBM-EC had significantly lower abundance of tight junction proteins (e.g., tight junction protein 1, 0.4-fold) and drug-metabolizing enzymes (e.g., glutathione-<i>S</i>-transferase A4, 0.4-fold) compared to control-EC, alongside an up- and down-regulation of drug transporters (e.g., long-chain fatty acid transport protein 4, 5-fold; adenosine triphosphate binding cassette transporter subfamily B member 1A, 0.3-fold). A large, 7-fold up-regulation of the endothelial cell surface receptor melanoma cell adhesion molecule (MCAM) and scavenger receptor class B member 1 (SCARB1) were identified in GBM-EC compared to control-EC. Immunohistochemistry confirmed cerebral endothelial localization of MCAM and SCARB1 in GBM, in addition to nonvascular patterning within the GBM, suggesting these receptors may be targets that could be exploited for drug delivery. The present study identified changes to BBB markers of paracellular permeability, as well as active and receptor-mediated transcellular transport that could present novel avenues to consider to enhance the permeability and GBM access of current and future therapeutics.</p>","PeriodicalId":52,"journal":{"name":"Molecular Pharmaceutics","volume":" ","pages":""},"PeriodicalIF":4.5,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145311958","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-Free-Based Thermophilic Biocatalyst for the Synthesis of Amino Acids from One-Carbon Feedstocks.","authors":"Ray Westenberg, Shaafique Chowdhury, Ryan Cardiff, Kimberly Wennerholm, Alexander S Beliaev, James M Carothers, Pamela Peralta-Yahya","doi":"10.1021/acssynbio.5c00352","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00352","url":null,"abstract":"<p><p>Bioproduction from one-carbon compounds, such as formate, is an attractive prospect due to reduced energy requirements and the possibility for using CO₂ as a sustainable feedstock. Formate-fixing pathways engineered using <i>Escherichia coli</i> lysate-based cell-free expression (CFE) biocatalysts have the potential to route 100% of feedstock carbon toward chemical synthesis but are undermined by siphoning of in-pathway metabolites and cofactors by the CFE background metabolism. To address this limitation, we engineer a CFE-based thermophilic multienzyme biocatalyst for the synthesis of serine and glycine from formate, bicarbonate, and ammonia. After expression of the thermophilic formate-to-serine pathway in a one-pot reaction, the mesophilic <i>E. coli</i> CFE background machinery is removed by simple heat denaturation, eliminating the siphoning of cofactors, in-pathway metabolites, and products. After bioprocess optimization, including pathway gene expression duration and chemical synthesis temperature, we achieve near stoichiometric conversion of formate and bicarbonate to serine and glycine, reaching 97% of stoichiometric yield. The use of a moderately thermophilic biocatalyst allowed chemical synthesis to take place at mesophilic temperatures, enabling the balance of optimal enzyme activity with minimal metabolite/cofactor thermal degradation. In a fed-batch experiment, the biocatalyst shows sustained chemical synthesis rates for 8 h, paving the way toward a continuous bioprocess. Finally, a sensitivity analysis of cofactor usage revealed that the most expensive cofactors, THF and NADPH, can be reduced by 5-fold without significantly lowering product yields. To the best of our knowledge, this is the first instance of expressing a thermophilic pathway in an <i>E. coli</i> lysate-based CFE system to generate a thermophilic biocatalyst for use at mesophilic temperatures. The CFE-based thermophilic formate-to-serine biocatalyst triples the combined serine and glycine yield previously obtained by a CFE-based mesophilic formate-to-serine biocatalyst (30%), and quadruple the yield obtained by a purified enzyme system (22%). Ultimately, this work opens the door to using <i>E. coli</i> lysate-based CFE for thermophilic biocatalyst generation to achieve high chemical synthesis yields.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure, Dynamics, and Interfacial Behavior in Ionic Liquid-Alcohol Binary Mixtures: A Molecular Dynamics Simulation Study.","authors":"Maryam Behzadi, Maryam Heydari Dokoohaki, Amin Reza Zolghadr","doi":"10.1021/acs.jpcb.5c05577","DOIUrl":"https://doi.org/10.1021/acs.jpcb.5c05577","url":null,"abstract":"<p><p>In this study, molecular dynamics (MD) simulations were conducted to elucidate the influence of cation type, anion type, and alcohol type on the bulk and interfacial properties of binary mixtures of ionic liquids (ILs) and alcohols. Simulations were performed at different mole fractions of ILs in the presence of methanol (MeOH) and butanol (BuOH). Four ILs were considered, comprising the cations 1,3-dimethylimidazolium ([MMIM]) and 1-butyl-3-methylimidazolium ([BMIM]) paired with the anions methyl sulfate ([MeSO₄]) and octyl sulfate ([OcSO₄]). The systems were examined in both the bulk phase and at the liquid-vapor interface. A range of analyses was employed to characterize structural, dynamics, and surface properties across mole fractions, including radial distribution functions, combined radial-angular distributions, density profiles, mean square displacements, and calculations of diffusion coefficients, surface tension, and molar electrical conductivity. The results show that the probability of cation-anion, cation-alcohol, and anion-alcohol interactions increases as the IL mole fraction decreases. The dominant hydrogen-bond interaction occurs between the oxygen atom of the sulfate anion and the hydroxyl hydrogen of the alcohol, with a closest distance of 0.18 nm and an angle of approximately 180°. Across all mole fractions, alcohol molecules exhibited higher mobility than the ionic species. Increasing the IL mole fraction resulted in a nonlinear decrease in the diffusion coefficients of all system components. Interfacial density profiles revealed that, at low mole fractions of [MMIM][MeSO₄] and [BMIM][MeSO₄], alcohols─particularly BuOH─are more prevalent in the vapor-phase region, while increasing IL concentration enriches the interface with ions. For [BMIM][OcSO₄], the anion consistently occupied the outermost layer toward the vapor phase at all concentrations. Surface tension variations with IL mole fraction were found to be strongly dependent on cation and anion identity. In the presence of MeOH, changes followed an approximately linear trend for all three ILs, whereas with BuOH, the trend was nonlinear, exhibiting a breakpoint associated with the onset of aggregation and micelle formation.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317953","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Concentration-Dependent Structures and Ultrafast Dynamics within Aqueous BeF₂: From Ion Complexes to Extended Networks.","authors":"Zhou Liang, Peng Jiahui, You Min, Yue Jianing, Fu XiaoBin, Shen Miao, Bian Hongtao, Qian Yuan","doi":"10.1021/acs.jpcb.5c04215","DOIUrl":"https://doi.org/10.1021/acs.jpcb.5c04215","url":null,"abstract":"<p><p>Salt-concentration effects on the structural dynamics of aqueous beryllium fluoride (BeF₂) must be elucidated to advance the separation and dehydration processes. This study systematically investigates BeF₂-solution concentration-dependent structures and dynamics using nuclear magnetic resonance and ultrafast spectroscopy. Be²⁺ primarily exists as the tetrahedral complex [BeF₂(H₂O)₂] in a BeF₂ solution, with small [BeF(H₂O)₃]⁺ and [BeF₃(H₂O)]^- complex contents. At BeF₂ concentrations exceeding 8.3 mol %, an extended Be-F network becomes prominent. Fourier transform infrared spectroscopy with a thiocyanate (SCN^-) vibrational probe reveals two SCN^- peaks: a bulk-water-associated peak and a Be²⁺-coordinated peak from the SCN^- substitution in [BeF_<i>x</i>(H₂O)₄-_<i>x</i>] complexes. The Be²⁺ peak full width at half-maximum exhibits nonlinear broadening at concentrations exceeding 8.3 mol %, consistent with network formation. Polarization-selective pump-probe measurements demonstrate that increased salt concentration accelerates vibrational energy relaxation and suppresses rotational diffusion. The rotational diffusion time is related to the macroscopic viscosity via the Stokes-Einstein-Debye model. The Be-F complexes cause non-Newtonian behavior of the BeF₂-solution viscosity. At concentrations exceeding 8.3 mol %, the formed network structures exponentially increase the viscosity, negating the classical Jones-Dole model. These macroscopic viscosity changes can be explained microstructurally. A coherent molecular mechanism linking the microscopic coordination structure to macroscopic transport properties is established.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"pH-Responsive Amylopectin Nanoparticles for On-Demand Glucose Production in Acidic Microenvironments.","authors":"Vinod Kumar Kannaujiya, Yijie Qiao, Peter R Wich","doi":"10.1021/acs.biomac.5c01095","DOIUrl":"https://doi.org/10.1021/acs.biomac.5c01095","url":null,"abstract":"<p><p>Glucose plays a crucial role in supplying energy to our bodies and fuels various cellular metabolic processes. An inadequate supply of glucose can disrupt these metabolic functions, potentially leading to health complications. To address these issues, we present a novel nanosystem designed to produce glucose in the slightly acidic environment found within the late endosomal/lysosomal compartment of cells. In this study, we employed amylopectin (AMY) polysaccharide as a substrate for the glucoamylase enzyme due to its biocompatible, biodegradable, and nontoxic nature. Through an acetal modification, amylopectin was transformed into an acid-sensitive hydrophobic material, acetalated amylopectin (AcAMY), enabling the formulation of particles and encapsulation of the glucoamylase with a high loading efficiency using a double emulsion method. These formulations showed a pH-dependent particle degradation and a controlled release of glucoamylase, facilitating the enzymatic hydrolysis of amylopectin to generate glucose. Moreover, this nanosystem exhibited efficient glucose production, reaching up to 80% glucose within 48 h under acidic conditions, in contrast to a maximum glucose production of 6% under physiological conditions. These findings demonstrate the particle's stability under the physiological environment of the bloodstream and highlight its ability to selectively produce glucose under acidic conditions. Cell viability results demonstrated that both enzyme-loaded and empty particles exhibit no toxicity, even at high particle concentrations, indicating excellent biocompatibility of this system. Consequently, this system shows great potential for effectively delivering glucose intracellularly to cells or tissues experiencing glucose deficiency.</p>","PeriodicalId":30,"journal":{"name":"Biomacromolecules","volume":" ","pages":""},"PeriodicalIF":5.4,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317929","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of Innovative Photosensitizers for Enhanced Photodynamic Therapy of Drug-Resistant Pathogens and Biofilms.","authors":"Hongshuang Qin, Yanxiang Guo, Chenjie Ma, Caihong Zhang, Ke Gao, Xueyi Sun, Chuanqi Zhao, Tao Liu","doi":"10.1021/acsinfecdis.5c00564","DOIUrl":"https://doi.org/10.1021/acsinfecdis.5c00564","url":null,"abstract":"<p><p>Antimicrobial photodynamic therapy (aPDT) has emerged as a potential approach to combating infections triggered by drug-resistant bacteria. However, the selection of photosensitizers for aPDT remains restricted, highlighting the urgent need to develop novel and efficient photosensitizers. Questiomycin A (QA) has garnered attention due to its diverse biomedical activities and good biocompatibility. However, the photodynamic effects of QA and its derivatives have not been explored. Investigating the photodynamic activities of QA-based compounds can provide innovative molecular structures for photosensitizer development. Herein, five derivatives (C1, C2, C3, C4, and C5) of QA are synthesized, and the aPDT activities of these compounds against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) are assessed. Our findings show that C5- and QA-mediated aPDT exhibits notable bactericidal efficacy, which is better than that of the well-known photosensitizer methylene blue. Especially C5 overcomes the drawbacks associated with the excitation of QA by blue-violet light, such as DNA damage and poor tissue penetration. Mechanism analyses reveal that C5-aPDT can induce singlet oxygen (¹O₂) generation to disrupt bacterial structures and functions, as well as eradicate biofilms and MRSA within biofilms. Studies in the mouse infection model indicate that C5-aPDT markedly promotes wound healing and exhibits excellent biocompatibility with a hemolysis rate of only 0.59% and minimal cell viability inhibition (2.96%) at therapeutic concentrations. To our knowledge, this is the first report that QA derivatives possess outstanding photodynamic activities, providing a unique molecular structure for the design of photosensitizers.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigating the Impact of Base Pair Mismatches on Cas13d Cleavage Efficiency Using Molecular Dynamics Simulations.","authors":"Ye Liu, Yan Li, Guohui Li, Huiying Chu","doi":"10.1021/acs.jpcb.5c04438","DOIUrl":"https://doi.org/10.1021/acs.jpcb.5c04438","url":null,"abstract":"<p><p>CRISPR-Cas13d enzyme has been transformed to an RNA-mediated tool for editing and manipulating nucleic acids, which has great promise in the field of genetic engineering. However, the presence of mismatches significantly undermines the cleavage efficiency of Cas13d to target RNA. The high sensitivity of Cas13d to base mismatches greatly limits its further application in related research areas such as nucleic acid testing and gene therapy. In this work, molecular dynamics simulations were employed to investigate the molecular mechanism of mismatches abolishing the activity of uncultured <i>Ruminococcus</i> sp. Cas13d (UrCas13d). Simulation results demonstrated that base pair mismatches of the target RNA were able to lead to an unwound opening and distortion of the Spacer:Target-RNA duplex, which enhances its interactions with the Helical-1 and Helical-2 domains of UrCas13d. Compared with the on-target system, the increase of those interactions caused by mismatches in mismatch systems hampered the conformation rearrangement of Helical-1 and Helical-2 to form an active conformation. Remarkably, the conformation rearrangement of the Helical-1 domain in mismatch systems also affects the relative position of residues in the HEPN-1 domain, particularly reflected in the movements of both residues Lys274-Phe292 and residues Asp311-Asn330 to residues Ala298-Asn308. Those movements reduced the steric hindrance effect of residues Lys274-Asn330 between residues Tyr212-Lys250 and residues Arg754-Lys772, which stimulated residues Tyr212-Lys250 moving close to residues Arg754-Lys772 in the HEPN-2 domain. The occurrence of this phenomenon resulted in the catalytic center burying into the hydrophobic interior of the UrCas13d protein, which could prevent the contact between the catalytic residues (R-X₄-H motifs) and the target RNA to decrease the cleavage efficiency of Cas13d protein to target RNA. The MD results reveal that blocking the transition of domains from inactive to active conformations and preventing the contact between R-X₄-H motifs and target RNA are the crucial determinants for mismatches to reduce UrCas13d activity. Those results contribute to providing theoretical support for the molecular mechanism of Cas13d that will stimulate future experimental research aimed at designing novel and efficient Cas13d variants to prevent undesired cleavages by regulating the interaction between nucleic acids and domains.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145317965","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanism and Free Energy Landscape of tRNA Charging at the Active Site of Leucyl tRNA Synthetase: A QM/MM Simulation Study with Enhanced Sampling.","authors":"Saheb Dutta, Nilashis Nandi, Amalendu Chandra","doi":"10.1021/acs.jpcb.5c04954","DOIUrl":"https://doi.org/10.1021/acs.jpcb.5c04954","url":null,"abstract":"<p><p>Leucyl-tRNA synthetase facilitates the transfer of leucine from leucyl-adenylate (Leu-AMP) to the 3'-terminal A76 of tRNA^<i>Leu</i> during the charging step of aminoacylation reaction. In the present study, we have investigated the charging step in leucyl tRNA synthetase from <i>E. coli (Ec)</i>. Utilizing hybrid QM/MM simulations and enhanced sampling techniques, we have explored the free energy landscape of the charging reaction. An analysis of the active site organization from μs long classical molecular dynamics simulations reveals the network of interactions essential for a catalytically competent conformation. The free energy profile obtained from QM/MM simulations unveils a two-step process where the amino group (NH₃⁺) of Leu-AMP serves as a general base. The first step of the charging process involves a proton transfer from O2' of A76 to the amino group of the leucyl-adenylate following the deprotonation of the positively charged amino group (NH₃⁺) to the carboxylic oxygen of Asp80, with a free energy barrier of 4.2 kcal mol^-1. In the second step, the deprotonated O2' attacks the carbonyl carbon (C) and breaks the C-O3α bond of the Leu-AMP. This step exhibits a free energy barrier of 22.9 kcal mol^-1 with a tetrahedral structure in the transition state. This study uncovers the charging pathway for leucyl tRNA synthetase in <i>E. coli</i>, with no water involvement in the charging step. It may be noted that the charging step varies between class I LeuRS (binding to 2'-OH of A76) and class II AspRS (binding to 3'-OH of A76). Despite these distinctions, both classes share a common pathway, highlighting the amino group's role as a base and the aspartate residue (Asp80 of LeuRS^<i>Ec</i> and Asp233 of AspRS^<i>Ec</i>) as the proton acceptor.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145306360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}