European Biophysics Journal最新文献

Association of a ligand with the binding site of a receptor is usually at least a two-step process - formation of an initial encounter complex followed by a conformational transition of the complex. Consequently, the description of binding by dimeric receptors requires a two-dimensional reaction scheme. An interesting example of a dimeric receptor is the decapping scavenger enzyme, DcpS. It is a critical determinant of mRNA metabolism that hydrolyses the 5’-end (hbox {m}^7)GpppN cap following 3’-end mRNA decay. The DcpS family of proteins function as homodimers with one active site in each protomer. We investigate the binding of substrate and product analogues of the mRNA cap, (hbox {m}^7)Gp((hbox {CH}_2))ppG and (hbox {m}^7)GMP, respectively, by human DcpS wild-type ((hbox {DcpS}^{mathrm {WT/WT}})) and its one-site compromised mutant ((hbox {DcpS}^{mathrm {WT/BC}})) using stopped-flow fluorimetry. Based on observations for the mutant (hbox {DcpS}^{mathrm {WT/BC}}), binding by each active site and for each ligand proceeds through the formation of an encounter complex followed by conformational transitions. In the case of (hbox {DcpS}^{mathrm {WT/WT}}), we show that only two association rate constants, one for the apo-enzyme with both sites empty and the second for the enzyme with one site already occupied, can be determined with satisfactory accuracy from experimental progress curves, even for experimental data with a high signal-to-noise ratio. An interesting and biologically relevant observation is that binding of substrate analogue by one site prevents binding by the remaining empty site, whereas in the case of the (hbox {m}^7)GMP product both sites bind ligand independently of the binding state of the other site.

The self-assembly of the cobaltabis(dicarbollide) (COSAN) anionic boron clusters into micelles above a critical micelle concentration (cmc) of 10–20 mM and its behavior as “sticky nano-ions” facilitating controlled protein aggregation have been previously investigated using scattering techniques. These techniques effectively provide average structural parameters but, when applied to colloidal systems, often rely on models assuming polydispersity or anisotropic shapes. Here, we employed sedimentation velocity analytical ultracentrifugation (SV-AUC), which offers the ability to resolve discrete species. We revisited two key questions: (1) the aggregation behavior of COSAN into micelles, a topic still under debate, and (2) the nature of the protein assemblies induced by COSAN, specifically their size/shape distribution and aggregation number. SV-AUC confirms the cmc of COSAN of 16 mM and reveals that COSAN micelles exhibit low aggregation numbers (8 in water and 14 in dilute salt), consistent with recent hypotheses. It shows that COSAN promotes myoglobin aggregation into discrete oligomeric species with well-defined aggregation numbers, such as dimers, tetramers, and higher-order assemblies, depending on the COSAN-to-protein ratio. COSAN binding could be quantified at the lower COSAN/myoglobin ratios. For example, at ratio 5, myoglobin monomer (25%) binds about two COSANs, dimer (45%) about 14 COSANs, and there are ≈ 30% very large aggregates. These results provide clarity on the discrete nature of COSAN micelle aggregation and protein assembly. This study highlights the complementary role of SV-AUC in understanding supramolecular assemblies, offering useful insights into the behavior of COSAN nano-ions and their interactions with biomacromolecules.

Biomembranes regulate molecular transport essential to cellular function and numerous biomedical applications, such as drug delivery and gene therapy. This study simulates molecular transport through nano-sized multipores in Giant Unilamellar Vesicles (GUVs) using COMSOL Multiphysics. We analyzed the diffusion dynamics of fluorescent probes—including Calcein, Texas-red dextran 3000 (TRD- 3k), TRD- 10k, and Alexa Fluor-labeled soybean trypsin inhibitor (AF-SBTI)—across different pore sizes, and derived rate constants using curve fitting that closely align with experimental data. Additionally, an analytical model based on Fick’s law of diffusion provides further insight into transport efficiency. This approach offers a novel perspective by examining simultaneous transport through multiple nanopores, which better mimics realistic biological environments compared to traditional single-pore studies. We used COMSOL for efficiently simulating large-scale, multi-nanopore systems, particularly in biomedical applications where modeling of complex transport phenomena is essential. This work provides new insights into multipore-mediated transport, critical for optimizing nanopore-based drug delivery and advancing the understanding of cellular transport mechanisms.

Water is central to biological processes not only as a solvent, but also as an agent shaping macromolecular behavior. Insights into water micro assemblies (WMA), defined by transient regions of low-density water (LDW) and high-density water (HDW), have highlighted their potential impact on biological phenomena. LDW, with its structured hydrogen bonding networks and reduced density, stabilizes hydrophobic interfaces and promotes ordered molecular configurations. Conversely, HDW, with its dynamic and flexible nature, facilitates transitions, solute mobility and molecular flexibility. By correlating experimental observations with simulations, we explore the influence of WMA on three key biological processes. In protein folding, LDW may stabilize hydrophobic cores and secondary structures by forming structured exclusion zones, while HDW may introduce dynamic flexibility, promoting the resolution of folding intermediates and leading to dynamic rearrangements. In enzyme catalysis, LDW may form structured hydration shells around active sites stabilizing active sites over longer timescales, while HDW may support substrate access and catalytic flexibility within active sites. In membrane dynamics, LDW may stabilize lipid headgroups, forming structured hydration layers that enhance membrane rigidity and stability, while HDW may ensure the nanosecond-scale flexibility required for vesicle formation and fusion. Across these tree processes, the WMA’s energy contributions, timescales and spatial scales align with the forces and dynamics involved, highlighting the role of LDW and HDW in modulating cellular interactions. This perspective holds implications for the design of lab-on-chip devices, advancements in sensor technologies, development of biomimetic membranes for drug delivery, creation of novel therapeutics and deeper understanding of protein misfolding diseases.

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 ((tau )) for VIEC single-exponential transients. Specifically, our results anticipate a power law dependence of (tau) 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.

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%.

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.