Protein Expression and Purification

Human Ca2+/calmodulin (CaM)-dependent protein kinase II alpha (CaMKII) is a highly abundant and conserved protein in the brain. It is a multifunctional serine/threonine kinase that phosphorylates cellular substrates across subcellular compartments, activating signaling pathways essential for long-term memory storage. However, a detailed understanding of protein structure and enzyme-substrate interaction mechanisms require a method to produce active, stable, and pure proteins at scale. Since phosphorylation can alter catalytic activity and substrate affinity, examining the non-phosphorylated state enables an unbiased characterization of basal enzymatic function. Therefore, producing the non-phosphorylated form of the kinase is important for biochemical and structural studies aimed at understanding kinase activation mechanisms. We describe a detailed protocol for the expression and high-yield purification of full-length, non-phosphorylated human CaMKII in bacterial cells. CaMKII was co-expressed with {lambda}-phosphatase to maintain it in a non-phosphorylated state. Protein expression was induced by autoinduction at low temperature to enhance the stability and solubility of CaMKII. Recombinant CaMKII was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKII has been considered difficult to purify in its non-phosphorylated form, we obtained it in high yield, and the protein was predominantly non-phosphorylated. Our results show that combining co-expression with autoinduction improves both the solubility and yield of the homogeneous non-phosphorylated form of CaMKII, which could be suitable for subsequent biochemical and structural studies.

Epigenetic regulation of chromatin dynamics via histone acetylation is one of several mechanisms by which eukaryotes regulate gene expression, DNA replication and repair, and maintain genome stability. This function is performed by histone acetyltransferases (HATs). Rtt109 is one such cytoplasmically localized HAT required for H3K56 acetylation found exclusively in fungi. Using recombinantly expressed Candida albicans Rtt109 and its chaperones, Vps75 and Asf1, we show that it can acetylate a 20-residue N-terminal H3 peptide in a coupled HAT assay only in the presence of Vps75, but not in the presence of Asf1 in vitro. This appears to be due to the fact that Rtt109-Vps75 is a high affinity stable complex, as estimated by biolayer interferometry (BLI) and gel filtration studies. The HAT activity of the Rtt109-Vps75 complex necessarily requires a flexible 118-160 residue loop of Rtt109 but not the C-terminal domain of Vps75. These results are comparable with what has been observed for the Saccharomyces cerevisiae Rtt109 homolog. In silico screening of 1,350,000 molecules from Life Chemicals Databases identified some likely inhibitors of C. albicans Rtt109 and six of them tested for binding to Rtt109 using BLI. The best ligand, F2368-0266, was used to study its effect on steady state enzyme kinetics, and found to be a competitive inhibitor of the peptide substrate but not of acetyl-CoA. Given the importance of Rtt109 in regulating virulence attributes such as hyphal morphogenesis and GPI biosynthesis in Candida albicans, and its effect on fungal pathogenesis, these results have significant clinical implications.

Background. Vaccines have been an essential tool in bringing the world close to polio eradication, with over 99.9 percent of the global population free of poliovirus. No antiviral drugs or monoclonal antibody products, however, are currently licensed for treatment of polio. We previously isolated a human monoclonal antibody (9H2) with potent neutralizing activity against all three poliovirus serotypes. To advance 9H2 as a therapeutic candidate, we optimized its sequence to extend serum half-life and improve manufacturability. Methods. A Multi-Attribute Method under stress conditions identified post-translational modification sites, and Abacus (Trademark) ranked sequence liabilities. Six amino acid substitutions were introduced into the variable regions, generating 23 combinatorial variants. Codon-optimized genes were synthesized and engineered into a human immunoglobulin G1 backbone containing crystallizable fragment (Fc) mutations (M428L/N434S) to extend serum half-life. Constructs were transfected into CHO K1 cells to generate stable pools in 24 well plates. Protein A purified antibodies were characterized using biophysical assays and an in vitro poliovirus neutralization assay. Results. All variants retained high in vitro neutralizing activity against poliovirus. A lead candidate was selected based on integrated assessment of biophysical properties across eight assays and expression yield. Conclusions. Structure-guided engineering and experimental evaluation enabled optimization of the 9H2 antibody sequence and identification of a lead candidate suitable for clinical manufacturing as a potential anti-poliovirus immunotherapeutic agent.

Drug targets and vaccines for bacterial diseases are challenging to develop. Computational subtractive genomics and reverse vaccinology offer a viable substitute for conventional drug and vaccine development approaches. In recent years, Pasteurella multocida, a Gram-negative pathogen responsible for severe infections in humans along with major diseases in livestock and poultry, has developed resistance to multiple drugs. As a result, there is an urgent demand for research into reliable, efficient, highly potent, and cost-effective strategies to combat the rise of Pasteurella multocida infections. To address this, the present study integrated an in silico subtractive genomics and reverse vaccinology workflow to pinpoint proteins with potential value as both drug and vaccine targets. From the complete Pasteurella multocida Pm70 proteome, we identified 446 proteins that had no similarities with human proteins, out of which 129 were found to be essential for the pathogens survival. These 129 proteins are therefore potential candidates for drug development. Further metabolic pathway analysis, virulence potential, subcellular localization, and structural analyses revealed two outer membrane proteins, namely, 47 kDa outer membrane protein and lipopolysaccharide-assembly protein LptD, as promising candidates for therapeutic development. Subsequent antigenicity analysis revealed both outer membrane proteins as potential vaccine targets, exhibiting highly favorable antigenicity scores. Candidate B- and T-cell epitopes were systematically mapped from these two targets based on antigenicity, immunogenicity, toxicity, and allergenicity potential and only high-confidence epitopes were assembled using adjuvants and class-specific linkers to construct the final multi-epitope vaccine (MEV). The designed MEV construct was demonstrated to be highly antigenic, non-toxic and non-allergenic to humans, while possessing favorable structural and physicochemical properties, and exhibited strong binding interactions with human toll-like receptors with minimal molecular deformability. Although further experimental validation is recommended to fully assess their efficacy, the present study establishes a solid foundation for the development of novel therapeutics and vaccine candidates against P. multocida, ultimately contributing to the reduction of its rising infections.

We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo. This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3{gamma} and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3{gamma}-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3{gamma}-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.

AbstractMetarhizium anisopliae is an important entomopathogenic fungi used in biological control of agricultural pests, but its commercial application is limited by relatively slow host mortality. This study aimed to engineer a chimeric protease (cPr1A) with enhanced binding affinity and protease activity against insect cuticle. We hypothesized that stronger cuticle binding would increase local enzyme concentration at the cuticle surface and thereby enhance cuticle degradation. To achieve this, the Bombyx mori chitin-binding domain (BmCBD) was fused to the C-terminus of the Pr1A protease from M. anisopliae. Recombinant Pr1A and cPr1A were expressed in Escherichia coli, purified by Ni-NTA affinity chromatography. Binding and protease activity were assayed in triplicate using Samia ricini cuticle powder as substrate. Results are presented as mean +/- SEM. The chimeric protease cPr1A showed a 28.9% increase in cuticle binding compared to wild-type Pr1A (15.81 +/- 1.97 vs. 12.27 +/- 2.13 g bound protein/mg cuticle powder; p < 0.002) and a 35% increase in protease activity (0.343 +/- 0.08 U/mg vs. 0.254 +/- 0.06 U/mg; p < 0.03). These results indicate that cPr1A is a promising candidate for overexpression in M. anisopliae to enhance cuticle degradation and potentially improve fungal virulence against insect pests.

Bacterial microcompartments (MCPs) are versatile proteinaceous organelles that compartmentalize metabolic pathways, offering promising scaffolds for synthetic biology and metabolic engineering. However, designing customized nanobioreactors requires distinguishing structurally indispensable shell proteins from those that can be modified or deleted to tune shell permeability without disrupting core organelle assembly. In this study, we performed a systematic biophysical and metabolic characterization of the hexameric shell protein PduU to evaluate its potential as a modular platform for synthetic organelle engineering. We tested whether deleting pduU or selectively truncating its N-terminal {beta}-barrel domain preserves shell assembly, metabolite flux, and intermediate confinement. Our results demonstrate that PduU modifications alter shell permeability while fully maintaining organelle structural integrity, monodispersity, and electrostatic colloidal stability. Crucially, this modulation in permeability redirects internal metabolic flux toward the energy-generating propionate pathway, resulting in elevated cell biomass and significantly increased yields of propionate, an economically vital industrial platform chemical. By establishing that PduU is a non-essential structural component whose modification tunes small-molecule flux, this work highlights PduU as a flexible locus for shell engineering, providing a scalable strategy for biomanufacturing of high-value bio-based products in tailor-made MCP nanobioreactors.

Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

Bordetella pertussis, the causative agent of whooping cough, is a reemerging public health threat. While the Tripartite Tricarboxylate Transporter (TTT) system BctCBA was previously implicated solely in citrate uptake, we demonstrate that the solute-binding protein BctC specifically binds citrate chelated with Zn{superscript 2} and Ni{superscript 2}. To elucidate the molecular mechanism of this interaction, we determined the crystal structures of BctC in three states: apo, open, and closed (citrate-zinc-bound), defining the structural determinants for metal-citrate recognition. Comparative analyses suggest that citrate-mediated divalent cation binding is a widespread feature among bacterial TTT homologs. Finally, in silico modeling of the full BctCBA complex predicts an elevator-type transport mechanism. Together, these findings redefine the functional scope of BctCBA, revealing a sophisticated strategy by which B. pertussis exploits organic chelators to acquire essential trace metals during infection.

Inhibin A and B (InhA and InhB) are potent negative regulators of follicle-stimulating hormone (FSH), which is essential for female fertility. InhA and InhB are heterodimeric transforming growth factor {beta} (TGF-{beta}) family ligands consisting of an inhibin (inh) subunit disulfide linked to an inhibin {beta}A or {beta}B (inh{beta}A, inh{beta}B) subunit, respectively. During purification, inhibins must be separated from activins (inh{beta}A or inh{beta}B homodimers), which always form when producing inhibins, and have the opposite effect on FSH synthesis. Current inhibin purification methods are technically challenging, require reagents that are not generally available, result in low purity or contamination with activins, or employ substitutions within the growth factor domain to favor inhibin formation over activin. We developed a simple and high-yielding expression and purification system for inhibins by transfection of suspension-cultured mammalian cells and tandem affinity chromatography, followed by reverse-phase purification of the mature ligands. We purified 1.4 mg (n = 3, SD = 0.47) and 0.093 mg (n = 3, SD = 0.043) of wild-type InhA and InhB per liter of culture medium with little to no contaminating activin A or B, at equal to higher purity and bioactivity than commercially available inhibins. Thus, we present a simple, robust, and versatile system for producing inhibins to interrogate their functions and mechanisms of action in vitro and in vivo.

With the rapid development of modern biotechnology, DNA vectors have become fundamental tools for inserting, transferring, and expressing specific gene sequences in various fields such as gene cloning, gene expression, gene editing, and gene therapy. However, when dealing with complex structured DNA sequences, traditional vector construction methods face challenges with low connection efficiency. This study proposes a new method for constructing recombinant vectors by employing a strategy of high-temperature treatment followed immediately by placement on ice, effectively reducing the complexity of DNA structures and enhancing the efficiency of PCR product-vector connection, thereby improving the construction efficiency of recombinant vectors. This paper describes the technical details of the method, experimental validation, and applications in gene cloning, gene recombination editing, and the preparation of gene therapy drugs, providing a new efficient tool for molecular biology experiments.

Anaerobic ammonium-oxidizing (anammox) bacteria are essential players in the global nitrogen cycle, responsible for converting ammonium and nitrite directly to nitrogen gas. Anammox bacteria have unique features such as a specialized cellular compartment - the anammoxosome. Candidatus (Ca.) Brocadia pituitae genome, as other anammox bacteria, encodes for a diversity of hydroxylamine oxidoreductase (HAO) paralogs, often pointed out as the enzymes responsible for most of the reactions of the anammox cycle. One of this Ca. B. pituitae HAO paralogs is an 840-amino acids protein, named here as BpMHAO, that stands out for its unprecedented domain organization, which includes a multicopper oxidase-like (MCo-like) domain followed by the HAO-like one. Sequence and structural analyses classified this MCo-like domain as homologous to the small laccase family. Spectroscopic characterization revealed a distinct UV-visible spectrum, tentatively assigned to the T3 center, whereas the EPR spectra confirmed the presence of T1, T2 and T3 copper centers. Enzymatic studies demonstrated limited laccase and oxygen-dependent ferroxidase activities. On the other hand, enzymatic assays performed in cell extracts from Escherichia coli and Shewanella oneidensis, harbouring the recombinant HAO-like domain, exhibited a robust hydroxylamine reductase activity using methyl viologen as the electron donor. Our results showed that the BpMHAO potentially plays a role in the anammox process/reactions by converting hydroxylamine into hydrazine. This feature can be relevant to anammox bacteria either by i) mitigating unwanted hydroxylamine, obtained by incorrect formation of this compound, by converting it into hydrazine and enabling its use in the anammox reaction or ii) using hydroxylamine from the outside medium as a substitute for ammonium, delivering hydrazine directly to the last step of the cycle, short-circuiting its first steps.

Circular single-stranded DNA (cssDNA) is a versatile biomolecule with applications spanning genome editing, DNA nanotechnology, synthetic biology, molecular diagnostics, and aptamer development. Compared with linear single-stranded DNA, cssDNA offers enhanced structural stability, resistance to in-cellulo degradation by exonucleases and enables the generation of long, sequence-defined DNA molecules that are difficult to obtain through conventional chemical synthesis methods. Despite its growing utility, widespread adoption of cssDNA has been limited by the lack of accessible, scalable, and cost-effective production methods, with many existing workflows relying on specialised reagents, extensive optimisation, or commercially synthesised DNA. Here, we present a streamlined, end-to-end protocol for the laboratory-scale production of high-purity cssDNA using an M13 phagemid-based system and standard molecular biology laboratory infrastructure. The workflow encompasses bacterial culture, phage amplification, nuclease treatment, phage precipitation, anion-exchange purification, and quality control, with practical optimisations to improve yield, reproducibility, and scalability. Using this approach, yields range from 120-195 {micro}g of purified cssDNA from 300 mL of culture supernatant. The protocol provides detailed guidance on critical process parameters, troubleshooting, and quality assessment, enabling reliable production of cssDNA suitable for a wide range of downstream molecular biology and genome engineering applications.

mRNA vaccines constitute a promising new platform for infectious disease prevention, having demonstrated their efficacy in response to the COVID-19 pandemic. While mRNA attributes such as purity, integrity, 5'-capping, polyA tail length, and nucleobase lipidation are critical to vaccine efficacy, another key attribute of any vaccine mRNA is its translatability - that is, how much antigen protein is produced by ribosomal translation. To assess mRNA translatability, cell-free translation (CFT) can be employed. Advantages of CFT include its rapid readout (~5 hours), minimal sample consumption, and measurement of antigen translation directly from mRNA, thereby eliminating any variability associated with cell transfection efficiency. This is in contrast to cell-based methods, which require larger amounts of mRNA material and formulation in lipid nanoparticles or lipofectamine to facilitate cell transfection. When developing a CFT method to assay mRNA translatability, one key consideration is which CFT system to use as there are several commercially available systems from a variety of different organisms and cell types. Presented here is the evaluation of three eukaryotic CFT systems - wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and HeLa cell lysate (HCL) - for the purpose of developing an analytical method to assay the translatability of mRNA. The dynamic range, linear range, sensitivity to thermal stress, and sensitivity to potential impurities (e.g., EDTA, double-stranded RNA) were determined for each system. Although HCL translated less protein than either WGE or RRL systems, it showed several advantages, notably a larger linear range and greater sensitivity to mRNA thermal stress and dsRNA impurities. Therefore, while it is still worthwhile to screen different CFT systems, the results presented here suggest that HCL should be strongly considered when developing an analytical method to assay translatability for mRNA vaccines.

Disintegrins are snake venom toxins that bind integrins and modulate platelet aggregation and cell migration. Isolation from venom often yields insufficient quantities for comprehensive study, making recombinant expression essential for detailed toxin characterisation. Here, we produced and characterised four disintegrins from Echis coloratus and Echis ocellatus in Escherichia coli. These homologous proteins contain distinct integrin-binding motifs: EcDis_RGD, EoDis_RGD, EcDis_KGD, and EcDis_VGD. Functional activities were evaluated using human platelet aggregation and endothelial cell scratch wound-healing assays. RGD-containing disintegrins exhibited the strongest inhibition of platelet aggregation, with EcDis_RGD showing the highest potency. The EcDis_KGD disintegrin inhibited platelet aggregation less potently, whereas EcDis_VGD showed no detectable activity in our assays. In endothelial cells, EcDis_RGD disintegrin markedly impaired cell migration, while EoDis_RGD displayed weaker anti-migratory activity. Neither KGD- nor VGD-containing disintegrins significantly affected wound closure. EoDis_RGD exhibited similar antiplatelet activity to the native, venom-purified disintegrin ocellatusin, validating our recombinant expression strategy. However, the corresponding PII-SVMP preparation displayed reduced inhibitory activity, consistent with incomplete generation of fully mature disintegrins species. Our findings demonstrate the importance of both integrin-binding motif identity and surrounding sequence context in determining disintegrin function, and support further use of recombinant toxins in toxinology and as therapeutic lead molecules.

Extracellular vesicles (EVs) are membrane-delimited nanoparticles, secreted by virtually all tested cell types to mediate intercellular and interorgan communication by transporting biomolecules, such as proteins, nucleic acids, and lipids, to recipient cells. Many EV isolation techniques have been developed for mammalian EVs and exploit specific EV properties, such as size, density, and solubility. Although Drosophila has emerged as a simple yet robust animal model for studying fundamental EV biology in its native context, it remains unclear whether commonly used EV isolation techniques can be applied to hemolymph (Drosophila blood). In this study, we first provide an in-depth characterization of particles in hemolymph using two complementary particle analysis techniques: dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). We then evaluate the performance of three commonly used small EV isolation methods--solvent precipitation, ultracentrifugation, and size-exclusion chromatography--for their ability to isolate small EVs from Drosophila larval hemolymph. All three methods can enrich small EVs from hemolymph to varying degrees, but none completely remove circulating proteins and lipoproteins. In particular, size-exclusion chromatography yields the purest small EV fractions, as evidenced by the enrichment of Drosophila orthologs of human small EV markers, including Tetraspanin 42Ee (Tsp42Ee), Tetraspanin 42Ed (Tsp42Ed), Tetraspanin 96F (Tsp96F), and Annexin B11 (AnxB11), although it produces the lowest protein yield. Altogether, our study provides practical guidance on both selecting an appropriate small EV isolation method and rigorously characterizing isolated small EV fractions according to specific research needs. More broadly, the knowledge gained from this study provides a framework for the isolation and characterization of small EVs in other insects, thereby facilitating future EV research across diverse insect species.

Laboratory automation is increasingly being adopted in academic research to improve throughput, reproducibility, and integration with computational workflows. A common bottleneck in molecular biology workflows is the isolation of DNA, typically performed with spin-column kits relying on centrifugation or vacuum. Existing automation solutions depend on magnetic separation systems requiring costly additional modules and specialist kits, making them impractical for laboratories that perform large numbers of extractions only occasionally. Here we present an open-source, 3D-printed vacuum manifold compatible with the Opentrons family of liquid handling robots that enables automated spin-column DNA isolation for up to 24 samples per run (96 with four manifolds in series). The manifold can be manufactured for approximately $2 using standard ABS filament and is compatible with widely available spin-columns, requiring no additional specialist reagents. Using this system, hands-on time for 24 plasmid minipreps was reduced by 80% compared to manual centrifugation and vacuum protocols. The automated workflow demonstrated comparable or greater plasmid yields and eliminated failed recoveries. All design files, labware definitions, and Opentrons protocols are released under the CERN Open Hardware Licence or MIT License. This device lowers the cost barrier to automated DNA purification for academic laboratories worldwide.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

Insect glutathione S-transferases (GSTs) play critical roles in xenobiotic detoxification and insecticide resistance, making them promising targets for selective pest-control strategies. Here, we performed a comparative analysis of GSTs representing multiple classes from beneficial insects, agricultural pests, and disease vectors. We found significant isozyme-specific variations in catalytic activity, stability, and conformational dynamics, notably, in relation to inhibition by the commercial chemical agents, ethacrynic acid (ECA) and permethrin (PER). Sequence similarity network analysis revealed distinct clustering of major GST classes and further highlighted the relatively recent evolutionary divergence of the insect-specific delta and epsilon classes. Structural modeling revealed highly conserved glutathione-binding sites (G-site), but substantial variation in the hydrophobic substrate-binding regions (H-site). Further, epsilon-class GSTs exhibited 4 helices oriented approximately 10{degrees} closer to the glutathione-binding site than delta enzymes, suggesting differences in active-site architecture. Steady-state kinetic analyses using 1-chloro-2,4-dinitrobenzene (CDNB) demonstrated that epsilon GSTs are generally more catalytically efficient. Inhibition studies revealed that ECA acts as a potent mixed-type inhibitor of delta-class GSTs, reducing catalytic efficiency by up to 56-fold, whereas PER produced weaker and more species-dependent effects. Notably, ECA binding strongly stabilized delta GSTs, as measured by differential scanning fluorimetry (DSF) and induced a selective rigidification of the 3/4 linker region and active-site motifs, as observed by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Collectively, these findings demonstrate the importance of analyzing the conformational dynamics mediating insect GST inhibition and provide a framework for exploiting isozyme-specific structural features in the design of next-generation selective insecticides.

The dCas9 system has rapidly been developed into many tools to explore different aspects of the human genome; the high binding specificity, coupled with the inactive nuclease enzyme, allows for precise recruitment of molecules to specific sequences of DNA. We sought to exploit these capabilities to create a tool for assessing the real-time proximity of two DNA sequences within a biological system. By incorporating aptamers into gRNAs, dCas9 molecules can be used to recruit the {beta}9 or {beta}10 strands of split-NanoLuc(R) to specific DNA sequences and quantify the proximity of those sequences based on their ability to complex with the luciferase fragment ({Delta}11S) and produce luminescence. While many tools exist to detect a single DNA sequence, this system is uniquely capable of assessing how two DNA sequences interact with each other. As expected, we found that the interaction of two dCas9 molecules was affected by their linear distance from each other on dsDNA. Surprisingly, we found that their interaction was also strongly influenced by rotational orientation, even for sequences that are close together in linear space. This finding indicates that dCas9 rotational alignment is an important consideration for designing dCas9 systems that target multiple DNA sequences simultaneously. Beyond the findings presented herein, we believe this DNA proximity detection tool has the potential to be adapted for applications involving the proximity and orientation of two DNA sequences.