Biophysical Analysis Research Articles

Eugenol and lidocaine inhibit voltage-gated Na+ channels from dorsal root ganglion neurons with different mechanisms.

Eugenol (EUG) is a bioactive monoterpenoid used as an analgesic, preservative, and flavoring agent. Our new data show EUG as a voltage-gated Na+ channel (VGSC) inhibitor, comparable but not identical to lidocaine (LID). EUG inhibits both total and only TTX-R voltage-activated Na+ currents (INa) recorded from VGSCs naturally expressed on dorsal root ganglion sensory neurons in rats. Inhibition is quick, fully reversible, and dose-dependent. Our biophysical and pharmacological analyses showed that EUG and LID inhibit VGSCs with different mechanisms. EUG inhibits VGSCs with a dose-response relationship characterized by a Hill coefficient of 2, while this parameter for the inhibition by LID is 1. Furthermore, in a different way from LID, EUG modified the voltage dependence of both the VGSC activation and inactivation processes and the recovery from fast inactivated states and the entry to slow inactivated states. In addition, we suggest that EUG, but not LID, interacts with VGSC pre-open-closed states, according to our data.

Prefoldins are novel regulators of the unfolded protein response in artemisinin resistant Plasmodium falciparum malaria

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug "Biperiden" that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive Pf3D7 and resistant Pf3D7k13R539T strains. Ring survival assays that are clinically relevant to analyze ART resistance in Pf3D7k13R539T parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PfPFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites.

Computational design of soluble and functional membrane protein analogues

De novo design of complex protein folds using solely computational means remains a substantial challenge1. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from G-protein-coupled receptors2, are not found in the soluble proteome, and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses demonstrate the high thermal stability of the designs, and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, as a proof of concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we have designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.

Aflatoxin biosynthesis regulators AflR and AflS: DNA binding affinity, stoichiometry, and kinetics.

Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared.

Structure and Substrate Recognition of the Escherichia coli Transport Protein NupG from the Nucleoside: H+ Symporter (NHS) Family

The nucleoside transporter NupG is one of the two principal transport proteins in the inner membrane of Escherichia coli that enable the organism to scavenge nucleosides from its external environment. NupG functions in a symport manner driven by the proton motive force and is a member of the Nucleoside:H+ Symporter (NHS) subfamily of the Major Facilitator Superfamily (MFS) of transporters. NupG has broad substrate specificity, transporting all naturally occurring purine and pyrimidine nucleosides. In studies over many years the nupG gene has been cloned and amplified, and the NupG protein has been purified, subjected to biochemical, biophysical and computational analysis, and its X-ray structure determined in the apo state at 3.0 Å resolution. The NupG structure had a typical MFS fold with twelve transmembrane spanning α-helices and distinct N- and C-terminal domains linked by a flexible loop. Preliminary site-directed mutagenesis and molecular docking studies on NupG identified nine putative nucleoside binding pocket residues (R136, T140, F143, Q225, N228, Q261, E264, Y318, F322) and a mutant (D323A) with 20-fold enhanced uridine binding activity. Further biochemical and structural investigations are necessary to better understand the substrate recognition and molecular mechanism of E. coli NHS family proteins (NupG, XapB, YegT).

Exploring the uncharted territory of the potential protein-protein interactions of cytosolic malate dehydrogenase.

In this review, we examine the protein-protein interactions of cytosolic malate dehydrogenase (MDH), an under-studied area in cellular metabolism. We provide a comprehensive overview of MDH involvement in metabolism, especially its interactions with metabolic partners and dynamics of changing metabolism. We present an analysis of the biophysical nature of these interactions and the current methods used to study them. Our review includes an assessment of computational docking studies, which offer initial hypotheses about potential MDH interaction partners. Furthermore, we provide a summary of the sparse yet insightful experimental evidence available, establishing a foundation for future research. By integrating biophysical analysis and methodological advancements, this paper aims to illuminate the intricate network of interactions involving cytosolic MDH and their metabolic implications. This work not only contributes to our understanding of MDH's role in metabolism but also highlights the potential impact of these interactions in metabolic disorders.

Biophysical and NMR analysis reveals binding affinity between HAX1 and CLPB proteins

HCLS1-associated protein X-1 (HAX1) is a multifunctional mitochondrial protein involved in the regulation of apoptosis, a crucial process of programmed cell death, and mRNA processing. Despite its significance, limited structural data is available for HAX1, hindering a comprehensive understanding of its biological function. Notably, the caseinolytic mitochondrial matrix peptidase chaperone subunit B (CLPB) has been identified as an interacting partner of HAX1, yet the biophysical properties and binding affinity governing their interaction remain poorly defined. In this study, we present a thorough biophysical characterization of full-length human HAX1 and CLPB, accomplished through recombinant expression and purification. By employing size exclusion chromatography, dynamic light scattering, and circular dichroism spectroscopy, we successfully established their biophysical properties, revealing contrasting structural features, with CLPB displaying α-helical content and HAX1 exhibiting a disordered nature. Moreover, we employed solution-state nuclear magnetic resonance (NMR) spectroscopy to probe their binding affinity. Our findings demonstrate the formation of stable multimeric complexes between HAX1 and CLPB, and we quantified a dissociation constant in the low range of micro-molar for their high affinity interaction. These results lay the foundation for further in-depth investigations into the dynamics and energetics governing the HAX1-CLPB interaction, ultimately contributing to a comprehensive understanding of their functional mechanisms.

Structural and biophysical analysis of cytochrome P450 2C9*14 and *27 variants in complex with losartan

The human cytochrome P450 (CYP) 1, 2 and 3 families of enzymes are responsible for the biotransformation of a majority of the currently available pharmaceutical drugs. The highly polymorphic CYP2C9 predominantly metabolizes many drugs including anticoagulant S-warfarin, anti-hypertensive losartan, anti-diabetic tolbutamide, analgesic ibuprofen, etc. There are >80 single nucleotide changes identified in CYP2C9, many of which significantly alter the clearance of important drugs. Here we report the structural and biophysical analysis of two polymorphic variants, CYP2C9*14 (Arg125His) and CYP2C9*27 (Arg150Leu) complexed with losartan. The X-ray crystal structures of the CYP2C9*14 and *27 illustrate the binding of two losartan molecules, one in the active site near heme and another on the periphery. Both losartan molecules are bound in an identical conformation to that observed in the previously solved CYP2C9 wild-type complex, however, the number of losartan differs from the wild-type structure, which showed binding of three molecules. Additionally, isothermal titration calorimetry experiments reveal a lower binding affinity of losartan with *14 and *27 variants when compared to the wild-type. Overall, the results provide new insights into the effects of these genetic polymorphisms and suggests a possible mechanism contributing to reduced metabolic activity in patients carrying these alleles.

Biophysical properties of alveolar surfactant in drever dogs with hunting associated pulmonary edema

BackgroundA syndrome of acute non-cardiogenic pulmonary edema associated with hunting is prevalent in the drever breed, but etiology of this syndrome is currently unknown. Alveolar surfactant has a critical role in preventing alveolar collapse and edema formation. The aim of this study was to investigate, whether the predisposition to hunting associated pulmonary edema in drever dogs is associated with impaired biophysical properties of alveolar surfactant. Seven privately owned drever dogs with recurrent hunting associated pulmonary edema and seven healthy control dogs of other breeds were included in the study. All affected dogs underwent thorough clinical examinations including echocardiography, laryngeal evaluation, bronchoscopy, and bronchoalveolar lavage (BAL) as well as head, neck and thoracic computed tomography imaging to rule out other cardiorespiratory diseases potentially causing the clinical signs. Alveolar surfactant was isolated from frozen, cell-free supernatants of BAL fluid and biophysical analysis of the samples was completed using a constrained sessile drop surfactometer. Statistical comparisons over consecutive compression expansion cycles were performed using repeated measures ANOVA and comparisons of single values between groups were analyzed using T-test.ResultsThere were no significant differences between groups in any of the biophysical outcomes of surfactant analysis. The critical function of surfactant, reducing the surface tension to low values upon compression, was similar between healthy dogs and affected drevers.ConclusionsThe etiology of hunting associated pulmonary edema in drever dogs is not due to an underlying surfactant dysfunction.

A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage by Candida albicans.

Candidalysin is a cytolytic peptide produced by the opportunistic fungal pathogen Candida albicans. This peptide is a key virulence factor in mouse models of mucosal and hematogenously disseminated candidiasis. Despite intense interest in the role of candidalysin in C. albicans pathogenicity, its host cell targets have remained elusive. To fill this knowledge gap, we performed a genome-wide loss-of-function CRISPR screen in a human oral epithelial cell line to identify specific host factors required for susceptibility to candidalysin-induced cellular damage. Among the top hits were XYLT2, B3GALT6 and B3GAT3, genes that function in glycosaminoglycan (GAG) biosynthesis. Deletion of these genes led to the absence of GAGs such as heparan sulfate on the epithelial cell surface and increased resistance to damage induced by both candidalysin and live C. albicans. Biophysical analyses including surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin physically binds to sulfated GAGs, facilitating its oligomerization or enrichment on the host cell surface. The addition of exogenous sulfated GAGs or the GAG analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate, but not non-sulfated dextran, also inhibited epithelial cell endocytosis of C. albicans and fungal-induced epithelial cell cytokine and chemokine production. In a murine model of vulvovaginal candidiasis, topical dextran sulfate administration reduced host tissue damage and decreased intravaginal IL-1β and neutrophil levels. Collectively, these data indicate that GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

Development of Peptide Displacement Assays to Screen for Antagonists of DDB1 Interactions.

The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-μM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.

Creating a more strategic small molecule biophysical hit characterization workflow

To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.

Fatty acid composition and biophysical characteristics of the cell membrane of feline spermatozoa

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

E-Cigarette Aerosol Alters Pulmonary Surfactant Function

Introduction: E-cigarette (EC) and vaping use continue to remain popular, particularly in young populations despite several clinical reports of vaping associated lung injury. One of the first compounds that EC aerosols comes into contact within the lungs during a deep inhalation is pulmonary surfactant. A complex mixture of lipids and proteins, surfactant reduces surface tension in the lungs maintaining compliance and preventing collapse. Currently, information on how EC aerosols impacts pulmonary surfactant remains limited, particularly in the in vivo scenario. We hypothesized that exposure to EC aerosol will impair the surface tension reducing ability of surfactant. Methods: In vitro EC aerosol exposure : Bovine Lipid Extract Surfactant (BLES) was used as a model surfactant in a direct exposure syringe system. BLES (2ml) was placed in a syringe (30ml) attached to an EC. The generated aerosol was drawn into the syringe and then expelled, repeated 30 times. In vivo EC aerosol exposure : Male and female Wistar rats were exposed to vehicle or menthol flavoured EC aerosol. Rats were exposed for 15 minutes for either a single or multiple (up to 8) exposures, with control animals receiving room air. Animals were euthanized and bronchoalveolar lavage was performed to collect surfactant. Analyzing surfactant function: Samples underwent dynamic compression and expansion, and biophysical analysis after exposure, completed using a constrained drop surfactometer (CDS). Results: Significant increases in minimum surface tensions were observed after exposure to EC aerosol across 20 compression/expansion cycles. Surfactant function was altered in vitro independent of vaping device, temperature, or inclusion of nicotine. Vehicle aerosol exposure produced alterations in surfactant properties, however addition of menthol flavouring significantly increased surface tensions compared to control and vehicle exposure. Preliminary in vivo studies indicate that aerosol exposure is well-tolerated by both male and female animals. Further experiments will analyze the function of surfactant isolated from these animals. Discussion: EC aerosols alter surfactant function through increases in minimum surface tension in vitro. This effect was more pronounced with menthol flavoured EC aerosol. It is anticipated that our in vivo experiments will corroborate these findings. Ultimately, impairment of surfactant due to EC aerosol exposure may contribute to lung dysfunction and susceptibility to further injury. Western University, NSERC, OGS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cold Spot SCANNER: Colab Notebook for predicting cold spots in protein–protein interfaces

BackgroundProtein–protein interactions (PPIs) are conveyed through binding interfaces or surface patches on proteins that become buried upon binding. Structural and biophysical analysis of many protein–protein interfaces revealed certain unique features of these surfaces that determine the energetics of interactions and play a critical role in protein evolution. One of the significant aspects of binding interfaces is the presence of binding hot spots, where mutations are highly deleterious for binding. Conversely, binding cold spots are positions occupied by suboptimal amino acids and several mutations in such positions could lead to affinity enhancement. While there are many software programs for identification of hot spot positions, there is currently a lack of software for cold spot detection.ResultsIn this paper, we present Cold Spot SCANNER, a Colab Notebook, which scans a PPI binding interface and identifies cold spots resulting from cavities, unfavorable charge-charge, and unfavorable charge-hydrophobic interactions. The software offers a Py3DMOL-based interface that allows users to visualize cold spots in the context of the protein structure and generates a zip file containing the results for easy download.ConclusionsCold spot identification is of great importance to protein engineering studies and provides a useful insight into protein evolution. Cold Spot SCANNER is open to all users without login requirements and can be accessible at: https://colab.research.google.com/github/sagagugit/Cold-Spot-Scanner/blob/main/Cold_Spot_Scanner.ipynb.

Characterization of G-quadruplex structures in genes involved in survival and pathogenesis of Acinetobacter baumannii as a potential drug target

Acinetobacter baumannii is a notorious pathogen that commonly thrives in hospital environments and is responsible for numerous nosocomial infections in humans. The burgeoning multi-drug resistance leaves relatively minimal options for treating the bacterial infection, posing a significant problem and prompting the identification of new approaches for tackling the same. This motivated us to focus on non-canonical nucleic acid structures, mainly G-quadruplexes, as drug targets. G-quadruplexes have recently been gaining attention due to their involvement in multiple bacterial and viral pathogenesis. Herein, we sought to explore conserved putative G-quadruplex motifs in A. baumannii. In silico analysis revealed the presence of eight conserved motifs in genes involved in bacterial survival and pathogenesis. The biophysical and biomolecular analysis confirmed stable G-quadruplex formation by the motifs and showed a high binding affinity with the well-reported G-quadruplex binding ligand, BRACO-19. BRACO-19 exposure also decreased the growth of bacteria and downregulated the expression of G-quadruplex-harboring genes. The biofilm-forming ability of the bacteria was also affected by BRACO-19 addition. Taking all these observations into account, we have shown here for the first time the potential of G-quadruplex structures as a promising drug target in Acinetobacter baumannii, for addressing the challenges posed by this infamous pathogen.

Delayed sowing and its ramifications: biophysical, yield and quality analysis of wheat cultivars in the northwest Indo-Gangetic plains.

The continuous cultivation of rice-wheat in the same field is a key element of double-cropping systems in the Indo-Gangetic plains. Yields of such cropping systems are increasingly challenged as climate change drives increases in temperature, terminal stress and uneven rainfall, delaying rice harvesting and subsequently delaying sowing of wheat. In this paper, we evaluate the optimum sowing dates to achieve high grain yield and quality of wheat cultivars in northwest India. Three cultivars of wheat, HD-2967, HD-3086 and PBW-723, were sown on three different dates at the research farm of ICAR-IARI, New Delhi, to generate different weather conditions at different phenological stages. Different biophysical attributes, photosynthetic rate, stomatal conductance and transpiration rate, were measured at different phenological stages. Yield and grain quality parameters such as protein, starch, amylopectin, amylose and gluten were measured in different cultivars sown on different dates. Biophysical parameters were found to be higher in timely sown crops followed by late-sown and very late-sown crops. Further, the different sowing dates had a significant (P < 0.05) impact on the grain quality parameters such as protein, starch, amylopectin, amylose and gluten content. Percentage increases in the value of starch and amylose content under timely sown were ~7% and 11.6%, ~5% and 8.4%, compared to the very late-sown treatment. In contrast, protein and amylopectin contents were found to increase by ~9.7% and 7.5%, ~13.8% and 16.6% under very late-sown treatment. High-temperature stress during the grain-filling periods significantly decreased the grain yield. Reduction in the grain yield was associated with a reduction in starch and amylose content in the grains. The protein content in the grains is less affected by terminal heat stress. Cultivar HD-3086 had higher growth, yield as well as quality parameters, compared to HD-2967 and PBW-723 in all treatments, hence could be adopted by farmers in northwest India. © 2024 Society of Chemical Industry.

Metal ion activation and DNA recognition by the Deinococcus radiodurans manganese sensor DR2539.

The accumulation of manganese ions is crucial for scavenging reactive oxygen species and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well-conserved domains - a DNA-binding domain, a dimerisation domain, and an ancillary domain - the mechanisms underlying both, metal ion activation and DNA recognition remain elusive. In this study, we present biophysical analyses and the structure of the dimerisation and DNA-binding domains of DR2539 in its holo-form and in complex with the 21 base pair pseudo-palindromic repeat of the dr1709 promoter region, shedding light on these activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein-DNA interfaces elucidates the significance of Tyr59 and helix α3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small-angle X-ray scattering experiments and supported by AlphaFold modeling provides a model illustrating the conformational changes induced upon metal binding.

Individual transcription factors modulate both the micromovement of chromatin and its long-range structure

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.

Evaluation of the cyclic single-chain Fv antibody derived from nivolumab by biophysical analyses and in vitro cell-based bioassay.

Single-chain Fv (scFv) is a recombinant small antibody in which a polypeptide linker connects the variable regions of the light chain (VL) and the heavy chain (VH). The practical use of scFv, however, has been prevented by its tendency to aggregate due to interchain VL-VH interactions. We recently developed a cyclic scFv whose N-terminus and C-terminus were connected by protein ligation techniques. Biophysical comparisons between cyclic and linear scFv have been conducted, but cell biological evaluations remain unexplored. Here we studied the properties of cyclic and linear scFv derived from nivolumab. Biophysical studies revealed that the thermal stability was not changed but that the antigen-binding activity was approximately 3-fold higher as a result of circularization. A cell-based PD-1/PD-L1 interaction inhibitory assay revealed that the biological activity of scFv was markedly higher in the circularized form. In addition, biophysical analysis of scFv proteins incubated in the presence of serum revealed that circularization suppressed the decrease in antigen-binding activity. It could be assumed that circularization of scFv improved stability in the presence of serum, which in turn would suggest the applicability of cyclic scFv as a biopharmaceutical format.