Pub Date : 2025-04-07DOI: 10.1007/s00249-025-01744-0
Sundeep Kapila, Pradeep R Nair
Exocytosis is a fundamental process related to the information exchange in the nervous and endocrine system. Among the various techniques, vesicle impact electrochemical cytometry (VIEC) has emerged as an effective method to mimic the exocytosis process and measure dynamic information about content transfer using nanoscale electrodes. In this article, through analytical models and large scale simulations, we develop scaling laws for the decay time constant for VIEC single-exponential transients. Specifically, our results anticipate a power law dependence of on the geometric and the transport parameters. This model compares very well with large scale simulations exploring the parameter space relevant for VIEC and with experimental results from literature. Remarkably, such physics-based compact models could allow for novel multi-feature-based self consistent strategies for back extraction of geometric and transport parameters and hence could contribute towards better statistical analysis and understanding of exocytosis transients and events.
{"title":"Extraction of geometric and transport parameters from the time constant of exocytosis transients measured by nanoscale electrodes.","authors":"Sundeep Kapila, Pradeep R Nair","doi":"10.1007/s00249-025-01744-0","DOIUrl":"https://doi.org/10.1007/s00249-025-01744-0","url":null,"abstract":"<p><p>Exocytosis is a fundamental process related to the information exchange in the nervous and endocrine system. Among the various techniques, vesicle impact electrochemical cytometry (VIEC) has emerged as an effective method to mimic the exocytosis process and measure dynamic information about content transfer using nanoscale electrodes. In this article, through analytical models and large scale simulations, we develop scaling laws for the decay time constant <math><mrow><mo>(</mo> <mi>τ</mi> <mo>)</mo></mrow> </math> for VIEC single-exponential transients. Specifically, our results anticipate a power law dependence of <math><mi>τ</mi></math> on the geometric and the transport parameters. This model compares very well with large scale simulations exploring the parameter space relevant for VIEC and with experimental results from literature. Remarkably, such physics-based compact models could allow for novel multi-feature-based self consistent strategies for back extraction of geometric and transport parameters and hence could contribute towards better statistical analysis and understanding of exocytosis transients and events.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143794312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-03DOI: 10.1007/s00249-025-01742-2
Greta Grassmann, Lorenzo Di Rienzo, Giancarlo Ruocco, Edoardo Milanetti, Mattia Miotto
Moving in a crowded cellular environment, proteins have to recognize and bind to each other with high specificity. This specificity reflects in a combination of geometric and chemical complementarities at the core of interacting regions that ultimately influences binding stability. Exploiting such peculiar complementarity patterns, we recently developed CIRNet, a neural network architecture capable of identifying pairs of protein core interacting residues and assisting docking algorithms by rescaling the proposed poses. Here, we present a detailed analysis of the geometric and chemical descriptors utilized by CIRNet, investigating its decision-making process to gain deeper insights into the interactions governing protein-protein binding and their interdependence. Specifically, we quantitatively assess (i) the relative importance of chemical and physical features in network training and (ii) their interplay at protein interfaces. We show that shape and hydrophobic-hydrophilic complementarities contain the most predictive information about the classification outcome. Electrostatic complementarity alone does not achieve high classification accuracy but is required to boost learning. Ultimately, our findings suggest that identifying the most information-dense features may enhance our understanding of the mechanisms driving protein-protein interactions at core interfaces.
{"title":"Exploring neural networks to uncover information-richer features for protein interaction prediction.","authors":"Greta Grassmann, Lorenzo Di Rienzo, Giancarlo Ruocco, Edoardo Milanetti, Mattia Miotto","doi":"10.1007/s00249-025-01742-2","DOIUrl":"https://doi.org/10.1007/s00249-025-01742-2","url":null,"abstract":"<p><p>Moving in a crowded cellular environment, proteins have to recognize and bind to each other with high specificity. This specificity reflects in a combination of geometric and chemical complementarities at the core of interacting regions that ultimately influences binding stability. Exploiting such peculiar complementarity patterns, we recently developed CIRNet, a neural network architecture capable of identifying pairs of protein core interacting residues and assisting docking algorithms by rescaling the proposed poses. Here, we present a detailed analysis of the geometric and chemical descriptors utilized by CIRNet, investigating its decision-making process to gain deeper insights into the interactions governing protein-protein binding and their interdependence. Specifically, we quantitatively assess (i) the relative importance of chemical and physical features in network training and (ii) their interplay at protein interfaces. We show that shape and hydrophobic-hydrophilic complementarities contain the most predictive information about the classification outcome. Electrostatic complementarity alone does not achieve high classification accuracy but is required to boost learning. Ultimately, our findings suggest that identifying the most information-dense features may enhance our understanding of the mechanisms driving protein-protein interactions at core interfaces.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143770838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-02DOI: 10.1007/s00249-025-01743-1
Eleonora Alfinito, Matteo Beccaria
The revival mechanism in dormant bacteria is a puzzling and open issue. We propose a model of information diffusion on a regular grid where agents represent bacteria and their mutual interactions implement quorum sensing. Agents may have different metabolic characteristics corresponding to multiple phenotypes. The intra/inter phenotype cooperation is analyzed under different metabolic and productivity conditions. We study the interactions between rapidly reproducing active bacteria and non-reproducing quiescent bacteria. We highlight the conditions under which the quiescent bacteria may revive. The occurrence of revival is generally related to a change in environmental conditions. Our results support this picture showing that revival can be mediated by the presence of different catalyst bacteria that produce the necessary resources.
{"title":"The sleeping bacterium: shedding light on the resuscitation mechanism.","authors":"Eleonora Alfinito, Matteo Beccaria","doi":"10.1007/s00249-025-01743-1","DOIUrl":"https://doi.org/10.1007/s00249-025-01743-1","url":null,"abstract":"<p><p>The revival mechanism in dormant bacteria is a puzzling and open issue. We propose a model of information diffusion on a regular grid where agents represent bacteria and their mutual interactions implement quorum sensing. Agents may have different metabolic characteristics corresponding to multiple phenotypes. The intra/inter phenotype cooperation is analyzed under different metabolic and productivity conditions. We study the interactions between rapidly reproducing active bacteria and non-reproducing quiescent bacteria. We highlight the conditions under which the quiescent bacteria may revive. The occurrence of revival is generally related to a change in environmental conditions. Our results support this picture showing that revival can be mediated by the presence of different catalyst bacteria that produce the necessary resources.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762573","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-30DOI: 10.1007/s00249-025-01741-3
John S Philo
The c(s) sedimentation distribution method implemented in the program SEDFIT (Biophys J 78:1606-1619, 2000) is widely used for analyzing sedimentation velocity data, and is particularly useful for detecting low levels of aggregates or other minor components in protein pharmaceuticals. Unfortunately, this method does not provide confidence limits for the area or sedimentation coefficient of each resolved peak, which makes it difficult to assess whether differences from one sample to another are statistically significant. This paper describes a new method to obtain such confidence limits using the program SVEDBERG (Biophys J 72:435-444, 1997) by automatically translating a saved c(s) distribution into a discrete species model where the molar masses of all species are constrained to keep the f/f0 ratio constant for all species. This approach also then allows relaxing the constant f/f0 ratio constraint on one or more minor species to determine their true molar masses (independent of assumptions about hydrodynamic shape), and also determining the confidence limits on that molar mass. It is demonstrated that this approach will work for samples containing up to five minor components (six total species), and even when multiple minor species are present at levels of only a few tenths of 1%.
{"title":"A method to determine confidence limits for the area, sedimentation coefficient, and molar mass of individual peaks from a SEDFIT c(s) distribution.","authors":"John S Philo","doi":"10.1007/s00249-025-01741-3","DOIUrl":"https://doi.org/10.1007/s00249-025-01741-3","url":null,"abstract":"<p><p>The c(s) sedimentation distribution method implemented in the program SEDFIT (Biophys J 78:1606-1619, 2000) is widely used for analyzing sedimentation velocity data, and is particularly useful for detecting low levels of aggregates or other minor components in protein pharmaceuticals. Unfortunately, this method does not provide confidence limits for the area or sedimentation coefficient of each resolved peak, which makes it difficult to assess whether differences from one sample to another are statistically significant. This paper describes a new method to obtain such confidence limits using the program SVEDBERG (Biophys J 72:435-444, 1997) by automatically translating a saved c(s) distribution into a discrete species model where the molar masses of all species are constrained to keep the f/f<sub>0</sub> ratio constant for all species. This approach also then allows relaxing the constant f/f<sub>0</sub> ratio constraint on one or more minor species to determine their true molar masses (independent of assumptions about hydrodynamic shape), and also determining the confidence limits on that molar mass. It is demonstrated that this approach will work for samples containing up to five minor components (six total species), and even when multiple minor species are present at levels of only a few tenths of 1%.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-26DOI: 10.1007/s00249-025-01740-4
Flora Fernandez-Sánchez, Jenny Flores-Ávila, Hugo S García, Edgar Mixcoha, Daniel Balleza
The bioactivity of the short antimicrobial peptides (ssAMPs) UyCT1, CT2, CT3, CT5, Uy17, Uy192, and Uy234 from the scorpion Urodacus yaschenkoi has been well-characterized. The antagonistic effect reported in those studies on some clinical isolates of pathogenic bacteria, including Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli was studied with an in silico approach to contrast their bioactivity in molecular terms. The peptides were modeled by generating high-quality structures with AlphaFold2, properly validated, and subjected to dynamic simulations in aqueous systems with the Gromos 43a1 and Charmm 36 force fields. Our analysis indicates that the degree of helicity of these peptides is closely linked to their composition and several physicochemical factors such as the hydrophobicity index, electrostatic potential, intrinsic flexibility, and dipole moment. We also found interesting parallels between the degree of order mentioned and the potency of each peptide with previously studied bacterial strains, specifically S. aureus. We analyzed in more detail of two specific peptides, UyCT1 and UyCT2, whose sequences are almost identical, except for the presence of a G-cap in the former. This subtle difference has a decisive impact on the conformational dynamics of these peptides, making the UyCT2 peptide more prone to disorder and the UyCT1 peptide more stable through the formation of multiple H-bonds. This analysis, based on an exhaustive characterization of the physicochemical properties of these ssAMPs, together with the determination of their conformational dynamics and the correlation with experimental data, could be the basis for the design and optimization of new drugs based on natural peptides found in scorpion venoms.
{"title":"Molecular dynamics study of the helix-to-disorder transition in short antimicrobial peptides from Urodacus yaschenkoi.","authors":"Flora Fernandez-Sánchez, Jenny Flores-Ávila, Hugo S García, Edgar Mixcoha, Daniel Balleza","doi":"10.1007/s00249-025-01740-4","DOIUrl":"https://doi.org/10.1007/s00249-025-01740-4","url":null,"abstract":"<p><p>The bioactivity of the short antimicrobial peptides (ssAMPs) UyCT1, CT2, CT3, CT5, Uy17, Uy192, and Uy234 from the scorpion Urodacus yaschenkoi has been well-characterized. The antagonistic effect reported in those studies on some clinical isolates of pathogenic bacteria, including Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli was studied with an in silico approach to contrast their bioactivity in molecular terms. The peptides were modeled by generating high-quality structures with AlphaFold2, properly validated, and subjected to dynamic simulations in aqueous systems with the Gromos 43a1 and Charmm 36 force fields. Our analysis indicates that the degree of helicity of these peptides is closely linked to their composition and several physicochemical factors such as the hydrophobicity index, electrostatic potential, intrinsic flexibility, and dipole moment. We also found interesting parallels between the degree of order mentioned and the potency of each peptide with previously studied bacterial strains, specifically S. aureus. We analyzed in more detail of two specific peptides, UyCT1 and UyCT2, whose sequences are almost identical, except for the presence of a G-cap in the former. This subtle difference has a decisive impact on the conformational dynamics of these peptides, making the UyCT2 peptide more prone to disorder and the UyCT1 peptide more stable through the formation of multiple H-bonds. This analysis, based on an exhaustive characterization of the physicochemical properties of these ssAMPs, together with the determination of their conformational dynamics and the correlation with experimental data, could be the basis for the design and optimization of new drugs based on natural peptides found in scorpion venoms.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143727135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-10DOI: 10.1007/s00249-025-01739-x
John S Philo
{"title":"Correction: SEDNTERP: a calculation and database utility to aid interpretation of analytical ultracentrifugation and light scattering data.","authors":"John S Philo","doi":"10.1007/s00249-025-01739-x","DOIUrl":"https://doi.org/10.1007/s00249-025-01739-x","url":null,"abstract":"","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584265","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-03DOI: 10.1007/s00249-025-01737-z
Marco A Saraiva
Currently, there is an increased interest in identifying the characteristics of amyloid aggregates in the initial stages of amyloid formation. The aggregation mechanism of the α-synuclein (Syn) amyloid protein, which has been extensively studied, is still not fully understood. I show that with conventional dynamic light scattering (DLS) technique, the measurements of the dimensions of Syn amyloid precursor forms can be done early in the protein incubation. Additionally, the early aggregation of the Syn protein was initially studied by analyzing autocorrelation functions from fit distributions up to 104 µs in the initial DLS measurements, specifically within the first 21 min. Investigation was conducted on the variation in the pH of the Syn solution throughout time. Based on DLS data, large Syn aggregated species formed from the monomer protein species. Afterward, I generated the autocorrelation functions based on the original DLS data, extending the fit distributions up to 105 µs and noticed the existence of elongated Syn amyloid precursor forms in the protein solutions. Because the length of the elongated Syn amyloid precursor forms closely matches the wavelength of the incident light, the combination of translational diffusion Dt and rotational diffusion Dr in the decay rates enabled the measurement of their geometric dimensions through DLS. The improved precision of the fitted distributions I offered resulted in a new interpretation for the Syn protein aggregation in the initial stages. Overall, the methodology used in this study could be an effective strategy for examining how Syn amyloid precursor forms develop over time.
{"title":"Determination of the size parameters of α-synuclein amyloid precursor forms through DLS analysis.","authors":"Marco A Saraiva","doi":"10.1007/s00249-025-01737-z","DOIUrl":"https://doi.org/10.1007/s00249-025-01737-z","url":null,"abstract":"<p><p>Currently, there is an increased interest in identifying the characteristics of amyloid aggregates in the initial stages of amyloid formation. The aggregation mechanism of the α-synuclein (Syn) amyloid protein, which has been extensively studied, is still not fully understood. I show that with conventional dynamic light scattering (DLS) technique, the measurements of the dimensions of Syn amyloid precursor forms can be done early in the protein incubation. Additionally, the early aggregation of the Syn protein was initially studied by analyzing autocorrelation functions from fit distributions up to 10<sup>4</sup> µs in the initial DLS measurements, specifically within the first 21 min. Investigation was conducted on the variation in the pH of the Syn solution throughout time. Based on DLS data, large Syn aggregated species formed from the monomer protein species. Afterward, I generated the autocorrelation functions based on the original DLS data, extending the fit distributions up to 10<sup>5</sup> µs and noticed the existence of elongated Syn amyloid precursor forms in the protein solutions. Because the length of the elongated Syn amyloid precursor forms closely matches the wavelength of the incident light, the combination of translational diffusion Dt and rotational diffusion Dr in the decay rates enabled the measurement of their geometric dimensions through DLS. The improved precision of the fitted distributions I offered resulted in a new interpretation for the Syn protein aggregation in the initial stages. Overall, the methodology used in this study could be an effective strategy for examining how Syn amyloid precursor forms develop over time.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
At present, research on the biomechanical response of the cupula of human semicircular canals (HSCs) has focused on indirect inference through the nystagmus view, which is limited by the participation of the human nervous system. In this study, 3D printing technology and hydrogel modification methods were used to fabricate a one-dimensional bionic semicircular canal (BSC) model with a ratio of 1:1 to the horizontal HSC. Target tracking technology was used to observe the deformation of the cupula. Then, constant angular acceleration stimulation and the other two stimulations were separately applied to the BSC to explore its biomechanical response. The results showed that the BSC had a similar time constant to that of the HSC, its maximum deviation displacement was proportional to the applied angular acceleration, and its amplitude-frequency gain under sinusoidal oscillation stimulation increased, but its phase difference decreased with increasing frequency, which consistent with the conclusions obtained by our theoretical deduction. The BSC model is expected to play a certain role in the mechanistic research and disease diagnosis of HSCs.
{"title":"Exploring the biomechanical response of human semicircular canals by a visualized bionic model.","authors":"Yani Jiang, Xianhua Wen, Guangcheng Xiang, Wenxuan Zhang, Junjie Dai, Junjie Gong, Yixiang Bian","doi":"10.1007/s00249-025-01738-y","DOIUrl":"https://doi.org/10.1007/s00249-025-01738-y","url":null,"abstract":"<p><p>At present, research on the biomechanical response of the cupula of human semicircular canals (HSCs) has focused on indirect inference through the nystagmus view, which is limited by the participation of the human nervous system. In this study, 3D printing technology and hydrogel modification methods were used to fabricate a one-dimensional bionic semicircular canal (BSC) model with a ratio of 1:1 to the horizontal HSC. Target tracking technology was used to observe the deformation of the cupula. Then, constant angular acceleration stimulation and the other two stimulations were separately applied to the BSC to explore its biomechanical response. The results showed that the BSC had a similar time constant to that of the HSC, its maximum deviation displacement was proportional to the applied angular acceleration, and its amplitude-frequency gain under sinusoidal oscillation stimulation increased, but its phase difference decreased with increasing frequency, which consistent with the conclusions obtained by our theoretical deduction. The BSC model is expected to play a certain role in the mechanistic research and disease diagnosis of HSCs.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-17DOI: 10.1007/s00249-025-01735-1
Alexander E Yarawsky, Judith A Ronau, Tiffany A Thibaudeau, Aaron C Ehlinger, Gekleng Chhor, Suki M Hyman, Michelle A Estrada, Vladimir Stojkovic, Michael T DeLion, Anil Vasudevan, Justin M Reitsma, Scott E Warder, Lake N Paul
Targeted protein degradation (TPD) has garnered appreciable interest in drug discovery due to its unique mechanism of action - degradation of a target in an event-driven manner, instead of traditional occupancy-driven inhibitor-based therapies. This is achieved by employing mono- or hetero-bifunctional small molecules known as degraders to induce the proximity of two proteins: a target protein and an E3 ubiquitin ligase, ultimately resulting in clearance of the target protein by the cell's inherent degradation machinery. A critical step in this pathway is ternary complex formation (TCF) between the ligase, degrader molecule, and the target protein. Although a bevy of biochemical, biophysical, cellular and structural approaches have been used to characterize degrader-induced ternary complexes, several knowledge gaps remain, such as stoichiometry and how much ternary complex is formed in solution. Analytical ultracentrifugation (AUC) is a biophysical method that is uniquely suited to address these questions, yet to this point has been surprisingly overlooked as an ideal method to characterize degrader candidates. In this study, we leveraged sedimentation velocity AUC (SV-AUC) to profile the degrader-induced ternary complex formation between Bruton's tyrosine kinase (BTK) and Cereblon (CRBN), allowing for evaluation of multiple attributes including sample purity, percent ternary complex, binding and kinetic rate constants, and hydrodynamics. We show that sedimentation equilibrium AUC (SE-AUC) can further complement the SV-AUC data with accurate molecular weight estimates of the ternary complex to confirm stoichiometry. This work demonstrates that AUC can be used both as a highly informative platform method for rapid characterization of candidate degrader compounds and as a rigorous method for elucidating additional details of the system.
{"title":"A paradigm shift: analytical ultracentrifugation as a multi-attribute platform method in targeted protein degradation.","authors":"Alexander E Yarawsky, Judith A Ronau, Tiffany A Thibaudeau, Aaron C Ehlinger, Gekleng Chhor, Suki M Hyman, Michelle A Estrada, Vladimir Stojkovic, Michael T DeLion, Anil Vasudevan, Justin M Reitsma, Scott E Warder, Lake N Paul","doi":"10.1007/s00249-025-01735-1","DOIUrl":"https://doi.org/10.1007/s00249-025-01735-1","url":null,"abstract":"<p><p>Targeted protein degradation (TPD) has garnered appreciable interest in drug discovery due to its unique mechanism of action - degradation of a target in an event-driven manner, instead of traditional occupancy-driven inhibitor-based therapies. This is achieved by employing mono- or hetero-bifunctional small molecules known as degraders to induce the proximity of two proteins: a target protein and an E3 ubiquitin ligase, ultimately resulting in clearance of the target protein by the cell's inherent degradation machinery. A critical step in this pathway is ternary complex formation (TCF) between the ligase, degrader molecule, and the target protein. Although a bevy of biochemical, biophysical, cellular and structural approaches have been used to characterize degrader-induced ternary complexes, several knowledge gaps remain, such as stoichiometry and how much ternary complex is formed in solution. Analytical ultracentrifugation (AUC) is a biophysical method that is uniquely suited to address these questions, yet to this point has been surprisingly overlooked as an ideal method to characterize degrader candidates. In this study, we leveraged sedimentation velocity AUC (SV-AUC) to profile the degrader-induced ternary complex formation between Bruton's tyrosine kinase (BTK) and Cereblon (CRBN), allowing for evaluation of multiple attributes including sample purity, percent ternary complex, binding and kinetic rate constants, and hydrodynamics. We show that sedimentation equilibrium AUC (SE-AUC) can further complement the SV-AUC data with accurate molecular weight estimates of the ternary complex to confirm stoichiometry. This work demonstrates that AUC can be used both as a highly informative platform method for rapid characterization of candidate degrader compounds and as a rigorous method for elucidating additional details of the system.</p>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-11DOI: 10.1007/s00249-025-01732-4
Sarah R. Dennison, Leslie H. G. Morton, Kamal Badiani, Frederick Harris, David A. Phoenix
The C-terminal amide carried by antimicrobial peptides (AMPs) can play a variable role in their antibacterial action and here, this role is investigated here for the synthetic peptide modelin-5 (M5-NH2). The peptide showed potent activity against Pseudomonas aeruginosa (MLC = 5.9 µM), with strong binding to the cytoplasmic membrane (CM) (Kd = 21.5 μM) and the adoption of high levels of amphiphilic α-helical structure (80.1%) which promoted strong CM penetration (9.6 mN m−1) and CM lysis (89.0%). In contrast, Staphylococcus aureus was resistant to M5-NH2 (MLC = 139.6 µM), probably due electrostatic repulsion effects mediated by Lys-PG in the organism’s CM. These effects promoted weak CM binding (Kd = 120.6 μM) and the formation of low levels of amphiphilic α-helical structure (30.1%), with low levels of CM penetration (4.8 mN m−1) and lysis (36.4%). C-terminal deamidation had a variable influence on the antibacterial activity of M5-NH2, and in the case of S. aureus, loss of this structural moiety had no apparent effect on activity. The resistance of S. aureus to M5-NH2 isoforms appeared to be facilitated by the high level of charge carried by these peptides, as well as the density and distribution of this charge. In the case of P. aeruginosa, the activity of M5-NH2 was greatly reduced by C-terminal deamidation (MLC = 138.6 µM), primarily through decreased CM binding (Kd = 118.4 μM) and amphiphilic α-helix formation (39.6%) that led to lower levels of CM penetration (5.1 mN m−1) and lysis (39.0%).
抗菌肽(AMPs)所携带的 C 端酰胺在其抗菌作用中可发挥不同的作用,本文对合成肽 modelin-5 (M5-NH2) 的这种作用进行了研究。该肽对铜绿假单胞菌(MLC = 5.9 µM)具有很强的活性,能与细胞质膜(CM)紧密结合(Kd = 21.5 μM),并具有高水平的两亲性α-螺旋结构(80.1%),能促进CM的穿透(9.6 mN m-1)和CM的裂解(89.0%)。相比之下,金黄色葡萄球菌对 M5-NH2 具有抗药性(MLC = 139.6 µM),这可能是由于该生物体 CM 中的 Lys-PG 介导的静电排斥效应。这些效应促进了微弱的 CM 结合(Kd = 120.6 μM),并形成了低水平的两亲α-螺旋结构(30.1%),具有低水平的 CM 穿透性(4.8 mN m-1)和裂解性(36.4%)。C 端脱氨对 M5-NH2 的抗菌活性有不同程度的影响,而对金黄色葡萄球菌来说,失去这一结构分子对活性没有明显影响。金黄色葡萄球菌对 M5-NH2 异构体的抗性似乎是由于这些肽所携带的高水平电荷以及电荷的密度和分布。就铜绿假单胞菌而言,M5-NH2 的活性因 C 端脱氨而大大降低(MLC = 138.6 µM),这主要是由于 CM 结合力降低(Kd = 118.4 μM)和两亲性 α-螺旋形成(39.6%)导致 CM 穿透力(5.1 mN m-1)和裂解率(39.0%)降低。
{"title":"The effect of C-terminal deamidation on bacterial susceptibility and resistance to modelin-5","authors":"Sarah R. Dennison, Leslie H. G. Morton, Kamal Badiani, Frederick Harris, David A. Phoenix","doi":"10.1007/s00249-025-01732-4","DOIUrl":"10.1007/s00249-025-01732-4","url":null,"abstract":"<div><p>The C-terminal amide carried by antimicrobial peptides (AMPs) can play a variable role in their antibacterial action and here, this role is investigated here for the synthetic peptide modelin-5 (M5-NH<sub>2</sub>). The peptide showed potent activity against <i>Pseudomonas aeruginosa</i> (MLC = 5.9 µM), with strong binding to the cytoplasmic membrane (CM) (K<sub>d</sub> = 21.5 μM) and the adoption of high levels of amphiphilic α-helical structure (80.1%) which promoted strong CM penetration (9.6 mN m<sup>−1</sup>) and CM lysis (89.0%). In contrast, <i>Staphylococcus aureus</i> was resistant to M5-NH<sub>2</sub> (MLC = 139.6 µM), probably due electrostatic repulsion effects mediated by Lys-PG in the organism’s CM. These effects promoted weak CM binding (K<sub>d</sub> = 120.6 μM) and the formation of low levels of amphiphilic α-helical structure (30.1%), with low levels of CM penetration (4.8 mN m<sup>−1</sup>) and lysis (36.4%). C-terminal deamidation had a variable influence on the antibacterial activity of M5-NH<sub>2</sub>, and in the case of <i>S. aureus</i>, loss of this structural moiety had no apparent effect on activity. The resistance of <i>S. aureus</i> to M5-NH<sub>2</sub> isoforms appeared to be facilitated by the high level of charge carried by these peptides, as well as the density and distribution of this charge. In the case of <i>P. aeruginosa,</i> the activity of M5-NH<sub>2</sub> was greatly reduced by C-terminal deamidation (MLC = 138.6 µM), primarily through decreased CM binding (K<sub>d</sub> = 118.4 μM) and amphiphilic α-helix formation (39.6%) that led to lower levels of CM penetration (5.1 mN m<sup>−1</sup>) and lysis (39.0%).</p></div>","PeriodicalId":548,"journal":{"name":"European Biophysics Journal","volume":"54 1-2","pages":"45 - 63"},"PeriodicalIF":2.2,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s00249-025-01732-4.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143389827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}