Scholarly Works, Biochemistry

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Identification and Mitigation of Inhibitory Substances Contained in High-Salinity Crude Glycerol Generated from Biodiesel Production for Polyhydroxyalkanoate Synthesis by Haloferax mediterranei
Zhang, Xueyao; Helm, Richard F.; McCoy, Emily L.; Zhao, Fujunzhu; Wang, Mingxi; Yebo Li, Stephanie Lansing; Huang, Haibo; Wang, Zhiwu (American Chemical Society, 2025-09-22)
High-salinity crude glycerol generated from biodiesel production poses significant challenges to microbial valorization due to inhibitory ingredients that severely limit microbial growth. This study identified and mitigated inhibitory substances contained in high-salinity glycerol sludge to enable its conversion to polyhydroxyalkanoates (PHAs) by the extreme halophilic archaeon Haloferax mediterranei. The long-chain fatty acids (LCFAs) were consistently identified as the primary inhibitors by liquid chromatography−mass spectrometry, Fourier transform infrared spectroscopy, and ultraviolet−visible spectroscopy. Acid precipitation at pH 2 efficiently removed these LCFAs, substantially reducing the required feedstock dilution from 23 to 3 times, improving PHA titer by 40%. Furthermore, this dilution reduction also increased the feedstock salinity utilization, achieving a 46% reduction in external salt supplementation for H. mediterranei growth. In contrast, overliming and arrested anaerobic digestion were confirmed to be ineffective in inhibitor removal. This study provides deep insights into inhibitor chemistry and presents acid precipitation as an effective pretreatment strategy for waste valorization of highsalinity crude glycerol.
How yoga interventions are operationalized and reported in the context of mental health and wellbeing RCTs: a systematic review and qualitative synthesis
Frazier, Mary C.; Remskar, Masha; Harden, Samantha M.; Barley, Karsen S.; David, Danielle E.; Guillen, Marina Z.; Olsen, Daryn E.; Markley, Kayla M.; Pullin, Megan J.; Brinsley, Jacinta (2025-12-22)
Background: Yoga is a popular intervention demonstrating promising impacts for mental health and wellbeing. Despite growing research interest, yoga remains poorly operationalized and inconsistently described in scientific literature, hindering dissemination, rigorous evaluation, and replication. This systematic review aims to address this critical knowledge gap by examining how yoga is operationalized in recent mental health and wellbeing research. Methods: We conducted a systematic review of literature from January 2013 to August 2024. Terms relating to yoga, mental health, wellbeing, and interventions were used to search MEDLINE, CINAHL, Embase, Emcare, PsycINFO, and Scopus. Randomized controlled trials that included yoga as the primary intervention and reported a validated measure of mental ill-health, mental wellbeing, or quality of life, were included. Inductive qualitative analyses of yoga definitions and descriptions were conducted. Results: Of 5206 studies identified, 129 were included with exclusion primarily due to study design. Qualitative analysis resulted in a total of 1291 meaning units (MU). Yoga definitions suggest that yoga is operationalized as a practice, complementary and alternative medicine, or system (e.g., encompassing philosophy and practices) with mind-body or mind-body-spirit aspects. Components of yoga included physical such as postures, mental such as meditation, and breath. Conclusions: This is the first systematic review to comprehensively analyze how yoga is operationalised and reported in recent experimental mental health and wellbeing research. Generally, yoga is operationalized as a mind-body or mind-body-spirit practice comprising mental, physical, and breathing components. We provide recommendations to improve the translation and implementation of yoga interventions. Trial registration This study was prospectively registered with PROSPERO (CRD42023455373). Clinical trial number: not applicable.
Cloning a codon-optimized version of the Rhagnium Inquisitor Antifreeze Protein gene into the pTYB12 expression vector
O'Donnell, Abby; Garrido, Josh; Mudumba, Rakshan; Hite, Kristopher (Elsevier, 2025-05)
The purpose of this project is to clone and express a synthetic version of antifreeze protein, RiAFP, from the arctic beetle, Rhagnium Inquisitor. The coding sequence for the RiAFP was codon-optimized for expression in E. Coli. Natively expressed RiAFP prevents the arctic beetle's body fluids from freezing by binding to the surface of ice crystals and inhibits the ability for the crystals to grow and expand.1 This protein exhibits a unique beta fold. The Beta sheets stack on top of each other in a repeating and parallel form. This protein is smaller than average as it has a size of 405 bp and a molecular weight of 13 kDA.2 It has been hypothesized that RiAFP may be applied to various cell-types in culture and improve cellular viability after the freezing process. So far, the codon-optimized version of the RiAFP gene has been successfully synthesized and amplified by PCR. The expected size of the amplicon, 405 bp, was verified with a 1 % agarose gel. After purification, the amplicon concentration was measured to be 263 ng/μL with a Qubit fluorimeter. The PCR product will be ligated into a pTYB12 expression vector backbone using HiFi assembly. Once successful cloning is confirmed via DNA sequencing the recombinant plasmid will be transformed into the BL21 expression strain of E. Coli. The RiAFP product will then be tested at various concentrations to test the hypothesis that inclusion in cryopreservation media will improve post thaw cellular viability. The data found from this project can hopefully be applied to the emerging field of cell therapy by investigating cellular freeze/thaw stress tolerance. 1.Lai Fun Kong, Ahmad Ayad Qatran Al-Khdhairawi, Bimo Ario Tejo; Rational design of short antifreeze peptides derived from Rhagium inquisitor antifreeze protein; Biocatalysis and Agricultural Biotechnology; Volume 23; 2020. 2.Hakim A, Nguyen JB, Basu K, Zhu DF, Thakral D, Davies PL, Isaacs FJ, Modis Y, Meng W; Crystal structure of an insect antifreeze protein and its implications for ice binding; J Biol Chem; Volume 17; 2013.
Genetically encoded chemical crosslinking of RNA in vivo
Sun, Wei; Wang, Nanxi; Liu, Hongjiang; Yu, Bingchen; Jin, Ling; Ren, Xingjie; Shen, Yin; Wang, Lei (Nature Portfolio, 2023-01)
Protein–RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N⁶-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N⁶-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein–RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics. [Figure not available: see fulltext.]
Genetically encoded chemical crosslinking of carbohydrate
Li, Shanshan; Wang, Nanxi; Yu, Bingchen; Sun, Wei; Wang, Lei (Nature Portfolio, 2023-01)
Protein–carbohydrate interactions play important roles in various biological processes, such as organism development, cancer metastasis, pathogen infection and immune response, but they remain challenging to study and exploit due to their low binding affinity and non-covalent nature. Here we site-specifically engineered covalent linkages between proteins and carbohydrates under biocompatible conditions. We show that sulfonyl fluoride reacts with glycans via a proximity-enabled reactivity, and to harness this a bioreactive unnatural amino acid (SFY) that contains sulfonyl fluoride was genetically encoded into proteins. SFY-incorporated Siglec-7 crosslinked with its sialoglycan ligand specifically in vitro and on the surface of cancer cells. Through irreversible cloaking of sialoglycan at the cancer cell surface, SFY-incorporated Siglec-7 enhanced the killing of cancer cells by natural killer cells. Genetically encoding the chemical crosslinking of proteins to carbohydrates (GECX-sugar) offers a solution to address the low affinity and weak strength of protein–sugar interactions. [Figure not available: see fulltext.]
Biochemical characterization of the self-sacrificing p-aminobenzoate synthase from Nitrosomonas europaea reveals key residues involved in selecting a Fe/Fe or Mn/Fe cofactor
Stone, Spenser; Peters, Logan; Fricke, Charlotte; Ray, W. Keith; Allen, Kylie D. (Springer, 2025-04)
A noncanonical route for p-aminobenzoate (pABA) biosynthesis in select bacteria utilizes a novel self-sacrificing heme oxygenase-like domain-containing oxidase/oxygenase (HDO) superfamily member. The recently characterized self-sacrificing pABA synthase from Chlamydia trachomatis (“CADD”) requires manganese and likely employs a heterobimetallic Mn/Fe cofactor. A conserved active site tyrosine residue is cleaved from the protein backbone to serve as the substrate for pABA synthesis and a lysine residue is the amino group donor. Here, we investigated the orthologous pABA synthase from the ammonia-oxidizing bacterium, Nitrosomonas europaea, which we refer to as NePabS. Consistent with the previously studied C. trachomatis enzyme, purified NePabS produces pABA in vitro in a reaction that only requires a metal cofactor, molecular oxygen, and a reducing agent, but no other substrates. Interestingly, maximal activity was observed with the addition of only iron as opposed to manganese and iron; thus, NePabS utilizes the more traditional Fe/Fe cofactor employed by most characterized HDO superfamily members. The self-sacrificing residues were confirmed to be Tyr25 and Lys159, which are the corresponding self-sacrificing residues in the CADD reaction. Strikingly, we could switch the metal dependence (Fe/Fe to Mn/Fe) and significantly improve the activity (~ twofold) of NePabS by substituting two phenylalanine residues with tyrosine residues (F148Y/F177Y), thus rendering the enzyme more similar to CADD. These results demonstrate that these two aromatic residues play an essential role in dictating metal specificity and potentially the proposed radical translocation process that facilitates the tyrosine cleavage reaction for pABA synthesis.
Habitat Disturbance Promotes Shifts in the Abundance of Major Fungal Phyla in the Roots of a Native Orchid, Tipularia discolor
Watkinson, Jonathan I. (Wiley, 2025-11-03)
Orchids are a widely distributed group of flowering plants with important roles in ecosystems around the globe. However, many species are in decline due, in part, to human-driven changes in their habitat. It is well established that orchids are reliant on specific groups of mycorrhizal fungi for growth and reproduction and that these fungi can vary across the range in which an orchid species resides. Recent studies have shown that the orchid fungal mycobiome (mycobiome) includes a diverse array of non-mycorrhizal endophytic fungi that may also contribute to growth and resilience and that can vary across a particular orchid's range. The communities of mycorrhizal and non-mycorrhizal species that make up the orchid mycobiome may be altered by habitat disturbance, which could affect the ability of these plant species to thrive in different environments. Here a metagenomic approach is used to provide a snapshot of the root mycobiome of Tipularia discolor in habitats defined as disturbed or undisturbed. While amplicon sequence variant (ASV) richness and evenness were similar, the structure of the mycobiome differed between the two sites. Orchids growing in disturbed locations were associated with a greater abundance of Basidiomycota and Glomeromycota, while orchids in undisturbed habitats were associated with Ascomycota and Mucoromycota. The overall abundance of mycorrhizal families was similar across the two habitats. The data indicate that habitat disturbance induces a change in the composition of the fungal mycobiome of T. discolor, suggesting that the community of root fungi could be key to the ability of orchids to successfully adapt to different environments.
Mushroom Tyrosinase Assay is used to Teach Enzyme Kinetics and Inhibition Studies in an Undergraduate Biochemistry Course
Unnoppet, Kylee; Hite, Kristopher (Wiley, 2022)
A spectrophotometric mushroom tyrosinase assay was optimized for use in the undergraduate biochemistry laboratory course at Virginia Tech. The laboratory exercise is designed to teach students about enzyme kinetics and tyrosinase‐inhibitor interactions. The tyrosinase protein structure was first studied using the python based molecular visualization software PyMOL and a PDB ID from the Protein Data Bank. The PDB ID that was used was PY9W and displays a H(2) L(2) tetramer. The H subunits of the complex contain a copper binding site that is coordinated by 3 histidine residues. The structure/function relationship was further studied using an immersive modeling hypercube. The assay uses white button mushrooms as the source of the tyrosinase enzyme, l‐3,4 dihydroxyphenylalanine (L‐DOPA) as the substrate, and kojic acid as the inhibitor. The optimal amounts of enzyme, substrate, and inhibitor are first determined, then a series of experiments are conducted which test varying concentrations of L‐DOPA against varying concentrations of kojic acid. Change in absorbance is measured at 475 nm in a spectrophotometer and the data is plotted to determine the kinetic constants and type of inhibition. Experimental kinetic constants are then compared to previously reported values. Further protein concentration in the tyrosinase extract is determined using Bradford assays. The incorporation of this exercise into the undergraduate biochemistry laboratory course at Virginia Tech will allow students to explore enzyme kinetics and tyrosinase‐inhibitor interactions.
Integration of experimental data and use of automated fitting methods in developing protein force fields
Polêto, Marcelo D.; Lemkul, Justin A. (2022-03)
The development of accurate protein force fields has been the cornerstone of molecular simulations for the past 50 years. During this period, many lessons have been learned regarding the use of experimental target data and parameter fitting procedures. Here, we review recent advances in protein force field development. We discuss the recent emergence of polarizable force fields and the role of electronic polarization and areas in which additive force fields fall short. The use of automated fitting methods and the inclusion of additional experimental solution data during parametrization is discussed as a means to highlight possible routes to improve the accuracy of force fields even further.
Monomeric and dimeric states of human ZO1-PDZ2 are functional partners of the SARS-CoV-2 E protein
Giacon, Noah; Lo Cascio, Ettore; Davidson, Darcy S.; Polêto, Marcelo D.; Lemkul, Justin A.; Pennacchietti, Valeria; Pagano, Livia; Zamparelli, Carlotta; Toto, Angelo; Arcovito, Alessandro (Elsevier, 2023-05)
The Envelope (E) protein of SARS-CoV-2 plays a key role in virus maturation, assembly, and virulence mechanisms. The E protein is characterized by the presence of a PDZ-binding motif (PBM) at its C-terminus that allows it to interact with several PDZ-containing proteins in the intracellular environment. One of the main binding partners of the SARS-CoV-2 E protein is the PDZ2 domain of ZO1, a protein with a crucial role in the formation of epithelial and endothelial tight junctions (TJs). In this work, through a combination of analytical ultracentrifugation analysis and equilibrium and kinetic folding experiments, we show that ZO1-PDZ2 domain is able to fold in a monomeric state, an alternative form to the dimeric conformation that is reported to be functional in the cell for TJs assembly. Importantly, surface plasmon resonance (SPR) data indicate that the PDZ2 monomer is fully functional and capable of binding the C-terminal portion of the E protein of SARS-CoV-2, with a measured affinity in the micromolar range. Moreover, we present a detailed computational analysis of the complex between the C-terminal portion of E protein with ZO1-PDZ2, both in its monomeric conformation (computed as a high confidence AlphaFold2 model) and dimeric conformation (obtained from the Protein Data Bank), by using both polarizable and nonpolarizable simulations. Together, our results indicate both the monomeric and dimeric states of PDZ2 to be functional partners of the E protein, with similar binding mechanisms, and provide mechanistic and structural information about a fundamental interaction required for the replication of SARS-CoV-2.
Membrane-localized MET engages PVR to mediate extranuclear juvenile hormone signaling in Aedes aegypti
Zhao, Wenhao; Griffith, Katara; Liang, Jiangtao; Dorodnitsyna, Maria; Liu, Pengcheng; Saunders, Thomas R.; Zhu, Jinsong (Proceedings of the National Academy of Sciences, 2025-11-18)
Although Methoprene-tolerant (MET) is well established as the intracellular receptor mediating the genomic actions of juvenile hormone (JH) in insects, the identity of the receptor responsible for initiating extranuclear JH responses has remained elusive. In the yellow fever mosquito Aedes aegypti, we identify the platelet-derived growth factor- and vascular endothelial growth factor-receptor related (PVR) protein as a key mediator of membrane-initiated JH signaling. JH treatment induces robust PVR phosphorylation in the fat body of adult female mosquitoes. Disruption of PVR function suppresses JH-induced activation of the phospholipase C and phosphatidylinositol 3-kinase signaling pathways and impairs primary follicle growth during the previtellogenic stage. Strikingly, JH-induced PVR phosphorylation and downstream signaling require MET, specifically its ligand-binding activity, but not its DNA-binding domain. A subpopulation of MET localizes to the plasma membrane of fat body cells, where it physically interacts with PVR between 24 h postemergence and 24 h post–blood feeding, suggesting that membrane-localized MET functions as the extranuclear JH receptor. Transcriptomic analyses following RNA interference (RNAi)-mediated knockdown of Met or Pvr demonstrate that PVR broadly contributes to JH-regulated gene expression. Notably, PVR-dependent signaling modulates genes that are also regulated by nuclear MET and enables JH to regulate additional gene sets independently of MET-mediated transcriptional regulation. These findings uncover a previously unrecognized MET–PVR signaling axis and support an integrated model of JH action in which membrane and nuclear pathways cooperate to enhance the specificity and complexity of JH function during the previtellogenic phase in mosquitoes.
CHARMM at 45: Enhancements in Accessibility, Functionality, and Speed
Hwang, Wonmuk; Austin, Steven L.; Blondel, Arnaud; Boittier, Eric D.; Boresch, Stefan; Buck, Matthias; Buckner, Joshua; Caflisch, Amedeo; Chang, Hao-Ting; Cheng, Xi; Choi, Yeol Kyo; Chu, Jhih-Wei; Crowley, Michael F.; Cui, Qiang; Damjanovic, Ana; Deng, Yuqing; Devereux, Mike; Ding, Xinqiang; Feig, Michael F.; Gao, Jiali; Glowacki, David R.; Gonzales, James E.; Hamaneh, Mehdi Bagerhi; Harder, Edward D.; Hayes, Ryan L.; Huang, Jing; Huang, Yandong; Hudson, Phillip S.; Im, Wonpil; Islam, Shahidul M.; Jiang, Wei; Jones, Michael R.; Kaser, Silvan; Kearns, Fiona L.; Kern, Nathan R.; Klauda, Jeffery B.; Lazaridis, Themis; Lee, Jinhyuk; Lemkul, Justin A.; Liu, Xiaorong; Luo, Yun; MacKerell Jr, Alexander D.; Major, Dan T.; Meuwly, Markus; Nam, Kwangho; Nilsson, Lennart; Ovchinnikov, Victor; Paci, Emanuele; Park, Soohyung; Pastor, Richard W.; Pittman, Amanda R.; Post, Carol Beth; Prasad, Samarjeet; Pu, Jingzhi; Qi, Yifei; Rathinavelan, Thenmalarchelvi; Roe, Daniel R.; Roux, Benoit; Rowley, Christopher N.; Shen, Jana; Simmonett, Andrew C.; Sodt, Alexander J.; Topfer, Kai; Upadhyay, Meenu; van der Vaart, Arjan; Vazquez-Salazar, Luis Itza; Venable, Richard M.; Warrensford, Luke C.; Woodcock, H. Lee; Wu, Yujin; Brooks, Charles L.; Brooks III, Bernard R.; Karplus, Martin (American Chemical Society, 2024-09-20)
Since its inception nearly a half century ago, CHARMM has been playing a central role in computational biochemistry and biophysics. Commensurate with the developments in experimental research and advances in computer hardware, the range of methods and applicability of CHARMM have also grown. This review summarizes major developments that occurred after 2009 when the last review of CHARMM was published. They include the following: new faster simulation engines, accessible user interfaces for convenient workflows, and a vast array of simulation and analysis methods that encompass quantum mechanical, atomistic, and coarse-grained levels, as well as extensive coverage of force fields. In addition to providing the current snapshot of the CHARMM development, this review may serve as a starting point for exploring relevant theories and computational methods for tackling contemporary and emerging problems in biomolecular systems. CHARMM is freely available for academic and nonprofit research at https://academiccharmm.org/program.
Drude polarizable force field for phosphorylated polypeptides and proteins
Gil Pineda, Laura I.; Miller, Candace J.; MacKerell, Alexander D. Jr.; Lemkul, Justin A. (Elsevier, 2025-10-22)
Phosphorylation is a ubiquitous posttranslational modification used across all domains of life to regulate protein stability, folding, subcellular localization, function, and activity. The most common targets of protein phosphorylation are the amino acids serine, threonine, and tyrosine. The considerable change in electrostatic character of these residues upon phosphorylation, from polar neutral to strongly negatively charged at physiological pH, implies a major change in biophysical properties of these residues. Modeling phosphorylated residues in molecular dynamics simulations is a challenge given their highly charged nature, and the development of polarizable force field parameters is an important task for the extension of protein force fields. Here, we present the parametrization and validation of force field parameters for phosphorylated serine, threonine, and tyrosine in all protonation states (neutral, monoanionic, and dianionic). We targeted a range of quantum mechanical properties for electrostatic and dihedral parameter fitting and subsequently validated the force field model in the context of two full-length proteins (ERK2 and WNK1) and short polypeptides. We found that the phosphorylated amino acids maintained the expected strong interactions with lysine and arginine residues, rigidifying loops in pERK2 and pWNK1. In the short polypeptides, phosphorylation of serine and threonine reduced disorder and promoted the formation of α-helical turns, in agreement with previous findings. The present force field is compatible with the Drude protein force field and expands the coverage of chemical space to include these important chemical entities for future investigations of protein structure and function, particularly for conformational changes that arise in intrinsically disordered proteins upon phosphorylation.
Robustness in biomolecular simulations: Addressing challenges in data generation, analysis, and curation
Brown, Anne M.; Lemkul, Justin A. (Cell Press, 2025-05-21)
Computational simulations of biomolecules provide a wealth of information about the thermodynamic landscape of biologically important systems, kinetics of important cellular processes, and the biophysical basis of life. Despite the ubiquity of molecular simulations in biophysical literature, major challenges persist for new practitioners entering the field, and even for experienced computational scientists, in maintaining and distributing their simulation outcomes. Here, we summarize critical obstacles encountered when performing biomolecular simulations and provide best practices for performing simulations that are robust, reproducible, and hypothesis-driven. We also discuss practices that promote improved reproducibility and accessibility using reliable tools and databases.
Running Gaussian-accelerated Molecular Dynamics Simulations in NAMD [Article v1.0]
Michel, Haley M.; Polêto, Marcelo D.; Lemkul, Justin A. (University of Colorado at Boulder, 2025-07)
Gaussian-accelerated molecular dynamics (GaMD) simulations are an advanced technique that enhances the sampling of configurational space by applying biasing potentials that reduce energy barriers, enabling faster exploration of the free energy landscape. This tutorial demonstrates the application of GaMD to the alanine dipeptide, serving as an accessible model system, and guides users through all GaMD simulation stages: conventional MD, GaMD equilibration, GaMD production, and reweighting. Users will gain practical insights into the preparation of input files, monitoring of GaMD convergence, and analysis of free energy profiles using PyReweighting. We make a particular effort to connect the underlying theory with the GaMD workflow. This tutorial is intended for users with prior molecular dynamics experience, Linux and command-line navigation, and with basic Python knowledge. The step-by-step instructions and accompanying scripts aim to streamline the GaMD workflow, making it accessible for the broader research community to explore enhanced sampling for a range of biomolecular systems.
Interface integrity in septin protofilaments is maintained by an arginine residue conserved from yeast to man
Grupp, Benjamin; Graser, Jano Benito; Seifermann, Julia; Gerhardt, Stefan; Lemkul, Justin A.; Gehrke, Jan Felix; Johnsson, Nils; Gronemeyer, Thomas (American Society for Cell Biology, 2025-05-01)
The septins are conserved, filament-forming, guanine nucleotide binding cytoskeletal proteins. They assemble into palindromic protofilaments which polymerize further into higher-ordered structures that participate in essential intracellular processes such as cytokinesis or polarity establishment. Septins belong structurally to the P-Loop NTPases but, unlike their relatives Ras or Rho, do not mediate signals to effectors through GTP binding and hydrolysis. Biochemical approaches addressing how and why septins utilize nucleotides are hampered by the lack of nucleotide-free complexes. Using molecular dynamics simulations, we determined structural alterations and intersubunit binding free energies in human and yeast septin dimer structures and in their in silico generated apo forms. An interchain salt bridge network around the septin unique β-meander, conserved across all kingdoms of septin containing species, is destabilized upon nucleotide removal, concomitant with disruption of the entire G-interface. Within this network, we confirmed a conserved arginine residue, which coordinates the guanine base of the nucleotide, as the central interaction hub. The essential role of this arginine for interface integrity was experimentally confirmed to be conserved in septins from yeast to human.
The pH-Dependent Specificity of Cathepsin S and Its Implications for Inflammatory Communications and Disease
DeHority, Riley; Gil Pineda, Laura I.; Cochran, Kari; Chen, Bentley; Bratek, Daniel; Helm, Richard F.; Lemkul, Justin A.; Zhang, Chenming (American Chemical Society, 2025-09-16)
Proteases have two major roles in health and disease: making functional changes to proteins as a post-translational modification and degradation of proteins as a regulatory or waste management mechanism. The cysteine protease cathepsin S serves both of these functions. It digests antigens in the adaptive immune system and is associated with many autoimmune diseases and cancers. Here, we show that the catalytic specificity of human cathepsin S is regulated by the pH conditions of its environment and identify the structural determinants of this switch. Peptide digests show that the proteolytic specificity of cathepsin S narrows at extracellular pH. Crystal structures reveal that a lysine residue descends into the S3 pocket of the active site above pH 7, which can be explained by changes in the protein's surface charge at that pH. We discuss biological compartment transitions and disease processes associated with cathepsin S in which these pH-dependent specificity switches may be triggered.
Weighted Ensemble Simulations With the Drude Polarizable Model
Polêto, Marcelo D.; da Silva, Gabriel Monteiro; Rubenstein, Brenda M.; Lemkul, Justin A. (Wiley, 2025-11-15)
Enhanced sampling methods have become powerful techniques to investigate complex systems and rare molecular events by facilitating greater access to configurational space. In parallel, the continuous development of polarizable force fields augments such techniques, allowing the investigation of the role of electronic polarization in transitional barriers that might be overlooked in nonpolarizable simulations. Together, improved sampling methods and polarizable force fields should accelerate computational discoveries. Here, we present an implementation of a weighted ensemble strategy for the Drude polarizable force field using the WESTPA software package in conjunction with the OpenMM simulation engine. We demonstrate its use for backbone conformational sampling in a model system (alanine dipeptide) and in studying functional sidechain rotations in an enzyme (Abl1 kinase). We further discuss possible consequences related to the inclusion of explicit electronic polarization in the model. This software implementation and validation should facilitate the use of WESTPA-Drude strategies throughout the simulation community.
A Molecular Biology-Based Practical Examination Used in a Capstone Laboratory Course for Biochemistry Majors
Hite, Kristopher (Elsevier, 2025-05)
A hands-on summative assessment was developed in a capstone laboratory course for biochemistry majors to evaluate students' ability to independently execute several molecular biological techniques simultaneously. These techniques included plasmid DNA quantification, assembling a series of restriction enzyme (RE) digests, loading an agarose gel, electrophoresis, imaging the resultant RE digest fragments, and finally, using deductive reasoning to interpret the banding pattern to draw a well-labeled restriction fragment map. A 3-page prompt was sent to students 48 hours prior to coming to the practical examination to provide time to prepare tables and protocols to successfully execute the experiments necessary for the restriction fragment map construction. Students were provided with a 200 ng aliquot of the pBR322 plasmid of unknown concentration. After using a Qubit fluorometer to determine DNA concentration students were told to use XmnI and BamHI REs to digest the plasmid and to save at least 40 ng of the original unknown plasmid aliquot to load on the agarose gel. Students were not told the exact identity of the plasmid nor were they told how many restriction digests to perform. The average time to complete this practical was approximately 3 hours and all students achieved interpretable gel image results. This exam was developed as an alternative to enzyme kinetics-based exams typically used in the same course for many years. All reagents necessary for this exam are commercially available making it easily adoptable.
Biogenic Amine-Containing 1,4-Naphthoquinones Mediate Extracellular Electron Transfer in Lactiplantibacillus plantarum
Blackburn, Benjamin T.; Barton, Joseph; Hoernig, Micah; Brown, Anne M.; Mevers, Emily (American Chemical Society, 2025-09-19)
Lactiplantibacillus plantarum, a lactic acid gut bacterium, uses exogenous quinones to facilitate extracellular electron transfer (EET) via type II NADH dehydrogenase (Ndh2). To probe Ndh2 specificity, we designed and evaluated a library of biogenic amine-substituted 1,4-naphthoquinones in an Ndh2-dependent EET assay. Analysis of mediator Ndh2 binding interactions revealed that activity correlates with key binding interactions. Specifically, mediators containing aromatic substitutions elicit favorable Ndh2 interactions, promoting EET.

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