Pub Date : 2025-10-29DOI: 10.1016/j.bpc.2025.107548
A. Pilarski , Gary A. Meints
The impact of solution conditions on the 31P isotropic chemical shifts of DNA phosphates and therefore the sequence-dependent backbone conformational equilibrium has not been well-documented. There are no previous studies of DNA backbone equilibrium in the presence of crowding agents, nor any on mismatched DNA. A systematic study of several experimental conditions (Na+ concentration, K+ concentration, Mg2+ concentration, pH, the presence of PEG molecular crowders) was performed and the effect quantified in mismatched DNA compared to a canonical control sequence. Na+ concentration, pH and crowding agents have only a minimal effect on the backbone equilibrium (<5 % perturbation on backbone populations). But in the mismatched DNA, both K+ and Mg2+ shift the backbone equilibrium on both DNA strands but most significantly perturb the phosphates in proximity to the mismatch. This indicates a possible role of counterions in mismatch recognition or nucleotide flipping, and suggests knowledge of solutions conditions continue to be relevant for conformational processes.
{"title":"Backbone equilibrium in mismatched DNA influenced by solution conditions","authors":"A. Pilarski , Gary A. Meints","doi":"10.1016/j.bpc.2025.107548","DOIUrl":"10.1016/j.bpc.2025.107548","url":null,"abstract":"<div><div>The impact of solution conditions on the <sup>31</sup>P isotropic chemical shifts of DNA phosphates and therefore the sequence-dependent backbone conformational equilibrium has not been well-documented. There are no previous studies of DNA backbone equilibrium in the presence of crowding agents, nor any on mismatched DNA. A systematic study of several experimental conditions (Na<sup>+</sup> concentration, K<sup>+</sup> concentration, Mg<sup>2+</sup> concentration, pH, the presence of PEG molecular crowders) was performed and the effect quantified in mismatched DNA compared to a canonical control sequence. Na<sup>+</sup> concentration, pH and crowding agents have only a minimal effect on the backbone equilibrium (<5 % perturbation on backbone populations). But in the mismatched DNA, both K<sup>+</sup> and Mg<sup>2+</sup> shift the backbone equilibrium on both DNA strands but most significantly perturb the phosphates in proximity to the mismatch. This indicates a possible role of counterions in mismatch recognition or nucleotide flipping, and suggests knowledge of solutions conditions continue to be relevant for conformational processes.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"329 ","pages":"Article 107548"},"PeriodicalIF":2.2,"publicationDate":"2025-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145463778","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
RufO is a Cytochrome P450 enzyme involved in synthesising Rufomycin, a circular peptide with antibacterial activity. Herein, we present structural and biophysical analyses to resolve the ambiguity of RufO's substrate specificity. The structure of unliganded RufO, alongside a series of computational and biophysical studies investigating its substrate specificity in the presence of ferredoxin, which is known to serve as an effector of the redox activities of several P450 enzymes. Contrary to reports on RufO's catalytic activity, monomeric L-tyrosine was not recognized by RufO in our isothermal titration calorimetry (ITC) experiments. Instead, RufO recognizes a range of putative substrates, particularly those containing methyl and nitro groups, suggesting a broader substrate scope. Additionally, we see that RufO binds to its redox partner CamB with micromolar affinity, and its interaction significantly enhances the putative substrate binding by ∼10-fold. Our crystal structure of RufO reveals similarities and differences in putative substrates and ferredoxin binding regions compared to other CYP450 enzymes. Our findings establish RufO might be a substrate-promiscuous enzyme with potential applications in the biocatalytic nitration of industrially relevant compounds.
{"title":"RufO, a cytochrome P450 (CYP) enzyme, recognition to putative substrates and a redox partner: Binding and structural insights","authors":"Dubey Saniya , Parate Shivani , Suman Abhishek , Vadivelu Abithaa , Priyanka Bajaj , Eerappa Rajakumara","doi":"10.1016/j.bpc.2025.107546","DOIUrl":"10.1016/j.bpc.2025.107546","url":null,"abstract":"<div><div>RufO is a Cytochrome P450 enzyme involved in synthesising Rufomycin, a circular peptide with antibacterial activity. Herein, we present structural and biophysical analyses to resolve the ambiguity of RufO's substrate specificity. The structure of unliganded RufO, alongside a series of computational and biophysical studies investigating its substrate specificity in the presence of ferredoxin, which is known to serve as an effector of the redox activities of several P450 enzymes. Contrary to reports on RufO's catalytic activity, monomeric L-tyrosine was not recognized by RufO in our isothermal titration calorimetry (ITC) experiments. Instead, RufO recognizes a range of putative substrates, particularly those containing methyl and nitro groups, suggesting a broader substrate scope. Additionally, we see that RufO binds to its redox partner CamB with micromolar affinity, and its interaction significantly enhances the putative substrate binding by ∼10-fold. Our crystal structure of RufO reveals similarities and differences in putative substrates and ferredoxin binding regions compared to other CYP450 enzymes. Our findings establish RufO might be a substrate-promiscuous enzyme with potential applications in the biocatalytic nitration of industrially relevant compounds.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"329 ","pages":"Article 107546"},"PeriodicalIF":2.2,"publicationDate":"2025-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145408282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-26DOI: 10.1016/j.bpc.2025.107547
Miriana Quaranta, Stefano Pascarella
Vitamin B6 and its vitamers are essential in bacteria. Many are auxotrophic for B6 vitamers and require salvage pathways and membrane uptake systems. Despite the importance of the uptake systems, very few transporters have been structurally characterized. The structure of the periplasmic binding protein (P5PA) of an ABC uptake transporter from the pathogen Actinobacillus pleuropneumoniae has been recently solved in complex with pyridoxal 5′-phosphate (PLP). Another close homolog from the same organism, AfuA, had been structurally characterized as a complex with glucose-6-phosphate (G6P). To study the molecular recognition of PLP by P5PA, a comparative approach has been applied. The heterologous complexes P5PA-G6P and AfuA-PLP have been generated by docking. Systematic molecular dynamics simulations have been applied to the native and heterologous complexes. Binding energies and molecular interactions have been compared for all the complexes. The results suggest the selective binding of the ligand is achieved by a combination of structural factors specific to each protein, including shape of the binding site, steric hindrance, hydrogen bonding, electrostatic and hydrophobic interactions. No single residue is uniquely responsible for ligand specificity although a few side chains play a significant role. Heterologous ligands are subject to destabilizing interactions that provoke the distortion of the ligand itself and the alteration of the protein dynamics. Residue Q267 appears to provide a significant stabilization contribution in the P5PA-PLP complex but not in AfuA-PLP. Likewise, D207 provides stabilization in the AfuA-G6P complex and not in AfuA-PLP. The indications obtained suggest strategies for the design of specific inhibitors.
{"title":"Structural basis of molecular recognition of pyridoxal 5′-phosphate in a bacterial periplasmic binding protein","authors":"Miriana Quaranta, Stefano Pascarella","doi":"10.1016/j.bpc.2025.107547","DOIUrl":"10.1016/j.bpc.2025.107547","url":null,"abstract":"<div><div>Vitamin B<sub>6</sub> and its vitamers are essential in bacteria. Many are auxotrophic for B<sub>6</sub> vitamers and require salvage pathways and membrane uptake systems. Despite the importance of the uptake systems, very few transporters have been structurally characterized. The structure of the periplasmic binding protein (P5PA) of an ABC uptake transporter from the pathogen <em>Actinobacillus pleuropneumoniae</em> has been recently solved in complex with pyridoxal 5′-phosphate (PLP). Another close homolog from the same organism, AfuA, had been structurally characterized as a complex with glucose-6-phosphate (G6P). To study the molecular recognition of PLP by P5PA, a comparative approach has been applied. The heterologous complexes P5PA-G6P and AfuA-PLP have been generated by docking. Systematic molecular dynamics simulations have been applied to the native and heterologous complexes. Binding energies and molecular interactions have been compared for all the complexes. The results suggest the selective binding of the ligand is achieved by a combination of structural factors specific to each protein, including shape of the binding site, steric hindrance, hydrogen bonding, electrostatic and hydrophobic interactions. No single residue is uniquely responsible for ligand specificity although a few side chains play a significant role. Heterologous ligands are subject to destabilizing interactions that provoke the distortion of the ligand itself and the alteration of the protein dynamics. Residue Q267 appears to provide a significant stabilization contribution in the P5PA-PLP complex but not in AfuA-PLP. Likewise, D207 provides stabilization in the AfuA-G6P complex and not in AfuA-PLP. The indications obtained suggest strategies for the design of specific inhibitors.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"329 ","pages":"Article 107547"},"PeriodicalIF":2.2,"publicationDate":"2025-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145399568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-21DOI: 10.1016/j.bpc.2025.107538
Erşed Akyüz , Mikhail M. Vorob'ev , Günnur Güler
Maintaining the structure and functionality of proteins is crucial in applications ranging from food preservation to pharmaceutical formulation. Ethanol, while commonly used as a solvent and preservative, can induce structural changes in proteins depending on its concentration and the specific structure of the protein itself. This study investigates the structural effects of ethanol on three types of model proteins, namely bovine serum albumin (BSA), β-Lactoglobulin (β-Lg), and β-Casein (β-Cn), by using UV–Vis spectroscopy and fluorescence spectroscopy. The conformational responses of proteins in water-EtOH solutions of various ethanol concentrations (0–10 %, v/v) were analyzed through absorbance and emission spectral changes. At increasing ethanol concentration, UV absorption data showed distinct protein-dependent spectral changes. β-Lg and β-Cn exhibited strong hypochromism (an absorbance decrease of ∼25 %) and red-shifting at 222 nm and 220 nm, respectively, indicating partial unfolding and solvent exposure of aromatic residues. BSA demonstrated subtle changes, and consistent quenching in fluorescence with a continuous blue-shifting to 330 nm, suggesting a moderate overall stability and local rearrangements in its structure. β-Cn exhibited red-shifted fluorescence and quenching, reflecting its flexible, disordered structure and heterogeneous response to solvent conditions. Statistical analysis revealed that while fluorescence spectroscopy was highly sensitive to the intrinsic differences between proteins (p < 0.001), the ethanol-induced conformational changes were too subtle to be detected as a statistically significant treatment effect. The consistency of these trends indicates a rational underlying mechanism of interaction. This reflects the subtle nature of the effect at the tested concentrations rather than the absence of an effect. Moreover, these results unveil the protein-specific effects of ethanol and strongly emphasize the importance of solvent composition in maintaining protein integrity. Ethanol concentrations up to 5 % may offer protein stability whereas high ethanol levels (≥ 5–10 %) promote structural perturbations. These results will be useful for both basic scientific research, such as biophysical studies and the advancement of optical techniques, and various industrial uses.
{"title":"Biophysical assessment of protein stability in ethanol-stressed environments via UV absorption and fluorescence spectroscopies","authors":"Erşed Akyüz , Mikhail M. Vorob'ev , Günnur Güler","doi":"10.1016/j.bpc.2025.107538","DOIUrl":"10.1016/j.bpc.2025.107538","url":null,"abstract":"<div><div>Maintaining the structure and functionality of proteins is crucial in applications ranging from food preservation to pharmaceutical formulation. Ethanol, while commonly used as a solvent and preservative, can induce structural changes in proteins depending on its concentration and the specific structure of the protein itself. This study investigates the structural effects of ethanol on three types of model proteins, namely bovine serum albumin (BSA), β-Lactoglobulin (β-Lg), and β-Casein (β-Cn), by using UV–Vis spectroscopy and fluorescence spectroscopy. The conformational responses of proteins in water-EtOH solutions of various ethanol concentrations (0–10 %, <em>v</em>/v) were analyzed through absorbance and emission spectral changes. At increasing ethanol concentration, UV absorption data showed distinct protein-dependent spectral changes. β-Lg and β-Cn exhibited strong hypochromism (an absorbance decrease of ∼25 %) and red-shifting at 222 nm and 220 nm, respectively, indicating partial unfolding and solvent exposure of aromatic residues. BSA demonstrated subtle changes, and consistent quenching in fluorescence with a continuous blue-shifting to 330 nm, suggesting a moderate overall stability and local rearrangements in its structure. β-Cn exhibited red-shifted fluorescence and quenching, reflecting its flexible, disordered structure and heterogeneous response to solvent conditions. Statistical analysis revealed that while fluorescence spectroscopy was highly sensitive to the intrinsic differences between proteins (<em>p</em> < 0.001), the ethanol-induced conformational changes were too subtle to be detected as a statistically significant treatment effect. The consistency of these trends indicates a rational underlying mechanism of interaction. This reflects the subtle nature of the effect at the tested concentrations rather than the absence of an effect. Moreover, these results unveil the protein-specific effects of ethanol and strongly emphasize the importance of solvent composition in maintaining protein integrity. Ethanol concentrations up to 5 % may offer protein stability whereas high ethanol levels (≥ 5–10 %) promote structural perturbations. These results will be useful for both basic scientific research, such as biophysical studies and the advancement of optical techniques, and various industrial uses.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"329 ","pages":"Article 107538"},"PeriodicalIF":2.2,"publicationDate":"2025-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145335032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-14DOI: 10.1016/j.bpc.2025.107537
Neha Devi , Satish Kumar Pandey , Nishima Wangoo
There has been a growing concern regarding antibiotic resistance which has led to the design of new antibacterial nanocomposite with high efficiency and low cytotoxicity. This report demonstrates an approach for the design of a nanocomposite comprising of histidine tagged silver nanoparticles (His@AgNPs) embedded it in a carboxymethyl chitosan hydrogel (CHG). Carboxymethyl chitosan was used as a cationic polymer substrate to reinforce the antibacterial activity of the embedded silver nanoparticles. ICP-MS showed sufficient release of silver ions which results in enhanced antibacterial activity. In addition, the hydrogel with embedded silver nanoparticles exhibited excellent antibacterial performance against gram-negative Escherichia coli and gram-positive Staphylococcus aureus through a “kill-release” stratagem, which was mainly due to the synergistic effect of CHG and Ag+ ions release. The results showed that the cross-linking may enhance antibacterial activities of nanocomposite (CHG-His@AgNPs) by approximately 4-folds in comparison to His@AgNPs. A strong signal observed in confocal microscopy confirmed the successful internalization of the nanocomposite in tested microorganisms. The change in the morphology of the bacterial cells upon the treatment with nanocomposite was observed through FE-SEM microscopy which confirmed the antibacterial activity of the nanocomposite. The synthesized nanocomposite can thereby serve as an excellent candidate for tackling antibiotic resistance.
{"title":"Cationic carboxymethyl chitosan nanofibers embedded with silver nanoparticles for enhanced antibacterial applications","authors":"Neha Devi , Satish Kumar Pandey , Nishima Wangoo","doi":"10.1016/j.bpc.2025.107537","DOIUrl":"10.1016/j.bpc.2025.107537","url":null,"abstract":"<div><div>There has been a growing concern regarding antibiotic resistance which has led to the design of new antibacterial nanocomposite with high efficiency and low cytotoxicity. This report demonstrates an approach for the design of a nanocomposite comprising of histidine tagged silver nanoparticles (His@AgNPs) embedded it in a carboxymethyl chitosan hydrogel (CHG). Carboxymethyl chitosan was used as a cationic polymer substrate to reinforce the antibacterial activity of the embedded silver nanoparticles. ICP-MS showed sufficient release of silver ions which results in enhanced antibacterial activity. In addition, the hydrogel with embedded silver nanoparticles exhibited excellent antibacterial performance against gram-negative <em>Escherichia coli</em> and gram-positive <em>Staphylococcus aureus</em> through a “kill-release” stratagem, which was mainly due to the synergistic effect of CHG and Ag<sup>+</sup> ions release. The results showed that the cross-linking may enhance antibacterial activities of nanocomposite (CHG-His@AgNPs) by approximately 4-folds in comparison to His@AgNPs. A strong signal observed in confocal microscopy confirmed the successful internalization of the nanocomposite in tested microorganisms. The change in the morphology of the bacterial cells upon the treatment with nanocomposite was observed through FE-SEM microscopy which confirmed the antibacterial activity of the nanocomposite. The synthesized nanocomposite can thereby serve as an excellent candidate for tackling antibiotic resistance.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"328 ","pages":"Article 107537"},"PeriodicalIF":2.2,"publicationDate":"2025-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318112","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cationic peptides have emerged as promising candidates in anticancer therapy due to their ability to directly target the plasma membrane of cancer cells, a mechanism that could potentially bypass traditional drug resistance pathways. In this study, we evaluated the cytotoxic activity and cell-membrane binding properties of three amphiphilic cationic peptides from the G(IIKK)ₙI-NH₂ family (n = 2–4) against human melanoma cells (SK-MEL-28). By performing MTT assays and tracking the propidium iodide (PI) uptake throughout peptide-cell interaction, we evaluated peptides' cytotoxicity. Assessment of the interaction dynamics was conducted by fluorescence spectroscopy assays with FPE, a surface potential sensitive probe. This evaluation indicated that an increase in net positive charge was relatable to a lower dissociation constant (Kd). Notably, G(IIKK)₄I-NH₂ showed the highest cytotoxicity, significant morphological alterations, rapid membrane permeabilization, and the lowest Kd, indicating a stronger membrane affinity when compared to the other peptides. G(IIKK)3I-NH₂, in the same manner as G(IIKK)₄I-NH₂, revealed a cooperative binding behavior, evidenced by a Hill coefficient > 1. An inverse correlation between peptide-cell membrane dissociation constants and cytotoxicity was established, supporting the notion that membrane interaction is a critical determinant of anticancer activity. In addition, we used a cell surface membrane potential probe to possibly anticipate the in vitro activity of cationic peptides. Altogether, these findings provide mechanistic insights into peptide-cell membrane interactions and may offer a basis for the rational design of novel anticancer peptides targeting melanoma.
{"title":"Probing the cytotoxicity and the dynamic interaction of IIKK cationic peptides with human melanoma cells","authors":"Luciana Marciano Sergio , Amanda Sansone Semerdjian , Manoel Arcisio-Miranda","doi":"10.1016/j.bpc.2025.107536","DOIUrl":"10.1016/j.bpc.2025.107536","url":null,"abstract":"<div><div>Cationic peptides have emerged as promising candidates in anticancer therapy due to their ability to directly target the plasma membrane of cancer cells, a mechanism that could potentially bypass traditional drug resistance pathways. In this study, we evaluated the cytotoxic activity and cell-membrane binding properties of three amphiphilic cationic peptides from the G(IIKK)ₙI-NH₂ family (<em>n</em> = 2–4) against human melanoma cells (SK-MEL-28). By performing MTT assays and tracking the propidium iodide (PI) uptake throughout peptide-cell interaction, we evaluated peptides' cytotoxicity. Assessment of the interaction dynamics was conducted by fluorescence spectroscopy assays with FPE, a surface potential sensitive probe. This evaluation indicated that an increase in net positive charge was relatable to a lower dissociation constant (K<sub>d</sub>). Notably, G(IIKK)₄I-NH₂ showed the highest cytotoxicity, significant morphological alterations, rapid membrane permeabilization, and the lowest K<sub>d</sub>, indicating a stronger membrane affinity when compared to the other peptides. G(IIKK)<sub>3</sub>I-NH₂, in the same manner as G(IIKK)₄I-NH₂, revealed a cooperative binding behavior, evidenced by a Hill coefficient > 1. An inverse correlation between peptide-cell membrane dissociation constants and cytotoxicity was established, supporting the notion that membrane interaction is a critical determinant of anticancer activity. In addition, we used a cell surface membrane potential probe to possibly anticipate the in vitro activity of cationic peptides. Altogether, these findings provide mechanistic insights into peptide-cell membrane interactions and may offer a basis for the rational design of novel anticancer peptides targeting melanoma.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"328 ","pages":"Article 107536"},"PeriodicalIF":2.2,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145311923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-11DOI: 10.1016/j.bpc.2025.107535
Katrina Brandmier, Kerney Jebrell Glover
Caveolin-1 (Cav1) is an integral membrane protein essential for the formation of caveolae, plasma microdomains implicated in signal transduction and mechanoprotection. Cav1 is comprised of three major alpha helices, but the topology these helices adopt remains unclear. Proline 110 is located between helix 1 and helix 2, and is hypothesized to enable Cav1 to adopt an intramembrane turn crucial for the cytosolic topology of Cav1. To assess the structural role of Proline 110, we utilized Förster resonance energy transfer (FRET) between native tryptophan (W128) and site-specifically labeled dansyl fluorophores to monitor conformational changes induced by the mutation of Proline 110 to Alanine (P110A). Static light scattering confirmed that all FRET constructs behaved monomerically, ensuring intramolecular energy transfer measurements. Our results show a significant decrease in FRET efficiency upon the P110A mutation consistent with a large conformational change. These findings support the critical role of P110 in maintaining the native topology of Cav1 and highlights the structural sensitivity of the intramembrane turn.
{"title":"Proline 110 is necessary for maintaining a compact helical arrangement in caveolin-1","authors":"Katrina Brandmier, Kerney Jebrell Glover","doi":"10.1016/j.bpc.2025.107535","DOIUrl":"10.1016/j.bpc.2025.107535","url":null,"abstract":"<div><div>Caveolin-1 (Cav1) is an integral membrane protein essential for the formation of caveolae, plasma microdomains implicated in signal transduction and mechanoprotection. Cav1 is comprised of three major alpha helices, but the topology these helices adopt remains unclear. Proline 110 is located between helix 1 and helix 2, and is hypothesized to enable Cav1 to adopt an intramembrane turn crucial for the cytosolic topology of Cav1. To assess the structural role of Proline 110, we utilized Förster resonance energy transfer (FRET) between native tryptophan (W128) and site-specifically labeled dansyl fluorophores to monitor conformational changes induced by the mutation of Proline 110 to Alanine (P110A). Static light scattering confirmed that all FRET constructs behaved monomerically, ensuring intramolecular energy transfer measurements. Our results show a significant decrease in FRET efficiency upon the P110A mutation consistent with a large conformational change. These findings support the critical role of P110 in maintaining the native topology of Cav1 and highlights the structural sensitivity of the intramembrane turn.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"328 ","pages":"Article 107535"},"PeriodicalIF":2.2,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145312022","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sodium dodecyl sulfate (SDS) is one of the most widely used detergents. Here, we discuss current knowledge regarding applications of SDS and its modes of interaction with proteins, particularly at low concentrations. SDS at 1–2 %, which is well above the critical micelle concentration, is commonly used to extract fully denatured and dissociated proteins and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in various applications, especially proteomics. In contrast, low concentration SDS may have been relatively underutilized. Here, we demonstrate the use of 0.1 % SDS for decellularization and protein fractionation. Why is 0.1 % SDS unique? The interaction between SDS and proteins is complex and depends on both the conditions and the proteins involved. At 0.1 %, the effects of SDS appear to be intermediate between negligible and extensive binding, highlighting its potential for novel applications. Two milder anionic detergents, Sarkosyl and sodium N-lauroyglutamate, whose effects are similar in certain applications to those of low concentration SDS, were briefly discussed.
{"title":"SDS protein interactions","authors":"Tsutomu Arakawa , Daisuke Ejima , Tomoto Ura , Teruo Akuta , Masamichi Oh-Ishi","doi":"10.1016/j.bpc.2025.107534","DOIUrl":"10.1016/j.bpc.2025.107534","url":null,"abstract":"<div><div>Sodium dodecyl sulfate (SDS) is one of the most widely used detergents. Here, we discuss current knowledge regarding applications of SDS and its modes of interaction with proteins, particularly at low concentrations. SDS at 1–2 %, which is well above the critical micelle concentration, is commonly used to extract fully denatured and dissociated proteins and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in various applications, especially proteomics. In contrast, low concentration SDS may have been relatively underutilized. Here, we demonstrate the use of 0.1 % SDS for decellularization and protein fractionation. Why is 0.1 % SDS unique? The interaction between SDS and proteins is complex and depends on both the conditions and the proteins involved. At 0.1 %, the effects of SDS appear to be intermediate between negligible and extensive binding, highlighting its potential for novel applications. Two milder anionic detergents, Sarkosyl and sodium N-lauroyglutamate, whose effects are similar in certain applications to those of low concentration SDS, were briefly discussed.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"328 ","pages":"Article 107534"},"PeriodicalIF":2.2,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145290894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-06DOI: 10.1016/j.bpc.2025.107533
Hari K.C. , Kumar Neupane , Khadka B. Chhetri , Rojina Ojha , Raj K. Acharya
Holy basil (Ocimum tenuiflorum) is primarily found in Nepal and India. In Ayurveda, it is commonly used as a traditional medicine to reduce pain, swelling, and various diseases. It has gained significant attention for its potential anti-inflammatory properties. One of the key targets associated with inflammation is Cyclooxygenase-2 (COX-2), an enzyme responsible for prostaglandin synthesis during the inflammatory response. In this study, we selected twenty flavonoids in the Holy Basil plant. These compounds were screened through Lipinski’s Rule of Five, followed by ADMET prediction. Virtual screening was conducted on the selected compounds against the COX-2 enzyme as a receptor using molecular docking techniques. Molecular docking study provides valuable insights at the molecular level into the interactions between Holy Basil compounds and COX-2. Furthermore, density functional computations were carried out utilizing the B3LYP method with the 6-311G basis, which is set to gain insight into the structural and electronic properties of the compounds. This study showcases the potential of flavonoids such as rhamnetin, Luteolin and kaempferol to act as anti-inflammatory agents, sparking further interest and research in this area.
圣罗勒（Ocimum tenuflorum）主要产于尼泊尔和印度。在阿育吠陀，它通常被用作一种传统药物来减轻疼痛、肿胀和各种疾病。它因其潜在的抗炎特性而受到广泛关注。与炎症相关的关键靶点之一是环氧化酶-2 (COX-2)，一种在炎症反应中负责前列腺素合成的酶。本研究从罗勒植物中提取了20种黄酮类化合物。这些化合物通过Lipinski 's Rule of Five进行筛选，然后进行ADMET预测。利用分子对接技术对选定的COX-2酶受体进行虚拟筛选。分子对接研究在分子水平上对罗勒化合物与COX-2的相互作用提供了有价值的见解。此外，利用6-311G基的B3LYP方法进行密度泛函计算，以深入了解化合物的结构和电子性质。这项研究展示了鼠李素、木犀草素和山奈酚等类黄酮作为抗炎剂的潜力，激发了这一领域的进一步兴趣和研究。
{"title":"Molecular docking and density functional theory studies of flavonoids of Holy basil plant against COX-2 enzyme","authors":"Hari K.C. , Kumar Neupane , Khadka B. Chhetri , Rojina Ojha , Raj K. Acharya","doi":"10.1016/j.bpc.2025.107533","DOIUrl":"10.1016/j.bpc.2025.107533","url":null,"abstract":"<div><div>Holy basil (Ocimum tenuiflorum) is primarily found in Nepal and India. In Ayurveda, it is commonly used as a traditional medicine to reduce pain, swelling, and various diseases. It has gained significant attention for its potential anti-inflammatory properties. One of the key targets associated with inflammation is Cyclooxygenase-2 (COX-2), an enzyme responsible for prostaglandin synthesis during the inflammatory response. In this study, we selected twenty flavonoids in the Holy Basil plant. These compounds were screened through Lipinski’s Rule of Five, followed by ADMET prediction. Virtual screening was conducted on the selected compounds against the COX-2 enzyme as a receptor using molecular docking techniques. Molecular docking study provides valuable insights at the molecular level into the interactions between Holy Basil compounds and COX-2. Furthermore, density functional computations were carried out utilizing the B3LYP method with the 6-311G basis, which is set to gain insight into the structural and electronic properties of the compounds. This study showcases the potential of flavonoids such as rhamnetin, Luteolin and kaempferol to act as anti-inflammatory agents, sparking further interest and research in this area.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"328 ","pages":"Article 107533"},"PeriodicalIF":2.2,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145263230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-26DOI: 10.1016/j.bpc.2025.107531
Michael T. Harnish, Bill Pham, Avery B. Arons, Yingjie Xu, Elias J. Fernandez, Tongye Shen
Nuclear receptors (NRs) are multidomain, ligand-activated transcription factors that play critical physiological roles. While the structured DNA-binding domain (DBD) and ligand-binding domain (LBD) have been well characterized, the function of intrinsically disordered regions—such as the hinge between the LBD and DBD—remains unclear. To illuminate the role of the hinge, we conducted five-microsecond molecular dynamics simulations of thyroid hormone receptor (TRα) alone versus bound to DNA. We reveal that DNA binding induces a significant structural change in the hinge region (helical to unwound coil), with a potentially important role in the regulation of TRα activity. Previously, hinge helicity has been reported to drive oligomerization and the consequent inhibition of coactivator binding, and such DNA-induced transition may promote TR activation. Protein-DNA binding is found to be multivalent and contains the direct interaction of the hinge with the DNA minor groove in addition to the canonical recognition helix of the DBD with the major groove. Furthermore, the poly-Arg segment of the hinge has a direct and significant influence on DNA conformation. This interaction promotes a bent DNA phosphate backbone, which might further contribute to the protein-DNA recognition. On a global scale, DNA binding induces a “closed-to-open” conformational change thus reducing direct DBD-LBD interactions, which corroborates previous calorimetric binding studies. Overall, our results provide insight into the mechanism of DNA recognition and the resulting conformational dynamics of the TRα-DNA complex.
{"title":"Conformational transition of a polycationic hinge domain contributes to DNA binding","authors":"Michael T. Harnish, Bill Pham, Avery B. Arons, Yingjie Xu, Elias J. Fernandez, Tongye Shen","doi":"10.1016/j.bpc.2025.107531","DOIUrl":"10.1016/j.bpc.2025.107531","url":null,"abstract":"<div><div>Nuclear receptors (NRs) are multidomain, ligand-activated transcription factors that play critical physiological roles. While the structured DNA-binding domain (DBD) and ligand-binding domain (LBD) have been well characterized, the function of intrinsically disordered regions—such as the hinge between the LBD and DBD—remains unclear. To illuminate the role of the hinge, we conducted five-microsecond molecular dynamics simulations of thyroid hormone receptor (TRα) alone versus bound to DNA. We reveal that DNA binding induces a significant structural change in the hinge region (helical to unwound coil), with a potentially important role in the regulation of TRα activity. Previously, hinge helicity has been reported to drive oligomerization and the consequent inhibition of coactivator binding, and such DNA-induced transition may promote TR activation. Protein-DNA binding is found to be multivalent and contains the direct interaction of the hinge with the DNA minor groove in addition to the canonical recognition helix of the DBD with the major groove. Furthermore, the poly-Arg segment of the hinge has a direct and significant influence on DNA conformation. This interaction promotes a bent DNA phosphate backbone, which might further contribute to the protein-DNA recognition. On a global scale, DNA binding induces a “closed-to-open” conformational change thus reducing direct DBD-LBD interactions, which corroborates previous calorimetric binding studies. Overall, our results provide insight into the mechanism of DNA recognition and the resulting conformational dynamics of the TRα-DNA complex.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"328 ","pages":"Article 107531"},"PeriodicalIF":2.2,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145263229","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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