European Biophysics Journal最新文献

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.

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) (K_d = 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 (K_d = 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 (K_d = 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%).

When bacteria interact with red blood cells, the plasma membrane receives signals from the microorganism adhesins, and the functional work of the erythrocyte as a whole depends on the biomembrane phospholipid condition. However, the microorganism effect on the structural and functional properties of the red blood cell membrane, as well as on the hemoglobin oxygen-binding ability has not been studied enough. Given the foregoing, we sought to study these issues in our work. The study used the "bacteria-red blood cells" model, using archival microbial strains (Staphylococcus aureus, Escherichia coli, Mycolicibacterium rutilum, and M. iranicum) and donor erythrocytes. The structural and functional properties of the red blood cell membrane phospholipids and the spectral characteristics of the hemoglobin molecule were studied using Raman spectroscopy. To study changes in red blood cell (RBC) morphology under the impact of microorganisms, laser interference microscopy was used. The results show that various types of microorganisms affected the conformational structure of the RBCs membrane phospholipid bonds, which contributed to changes in the morphological characteristics of cells, resulting in functional changes in both the red blood cell as a whole and the main RBC oxygen transport protein-hemoglobin.

Here we advance in the understanding of nucleic acids interactions with small anionic ligands by characterizing the binding of the Orange G (OG) dye to double-stranded DNA via single molecule force spectroscopy. While there is no detectable interaction at low ionic strengths, we found that for [ ${\text{Na}}^{+}$ ] = 150 mM OG was able to interact with the double-helix via groove binding in a non-cooperative way, with a relatively high equilibrium association constant ( $\sim$ ${10}^5$ ${\text{M}}^{-1}$ ) that is compatible to other classic DNA small ligands. Furthermore, experiments performed with a fixed OG concentration at various ionic strengths clearly show that the binding can be turned "on / off" by regulating the concentration of available counterions, a result that can guide the development of new synthetic ligands and shows how to modulate their interactions with nucleic acids. The present work therefore advances in evaluating the fundamental role of the ionic strength on the DNA interactions with small anionic ligands.

X-ray crystallography has tremendously served structural biology by routinely providing high-resolution 3D structures of macromolecules. The extent of information encoded in the X-ray crystallography is proportional to which resolution the crystals diffract and the structure can be refined to. Therefore, there is a continuous effort to obtain high-quality crystals, especially for those proteins, which are considered difficult to crystallize into high-quality protein crystals of suitable sizes for X-ray crystallography. Efforts in enhancing the resolution in X-ray crystallography have also been made by optimizing crystallization protocols using external stimuli such as an electric field and magnetic field during the crystallization. Here, we present the feasibility of on-the-fly post-crystallization resolution enhancement of the protein crystal diffraction by applying a high-voltage electric field. The electric field between 2 and 11 kV/cm, which was applied after mounting the crystals in the beamline, resulted in the enhancement of the resolution. The crystal diffraction quality improved progressively with the exposure time. Moreover, we also find that upto defined electric field threshold, the protein structure remains largely unperturbed, a conclusion further supported by molecular dynamics simulations.

Compared to fluorescence, second harmonic generation (SHG) has recently emerged as an excellent signal for imaging probes due to its unmatched advantages in terms of no photobleaching, no phototoxicity, no signal saturation, as well as the superior imaging accuracy with excellent avoidance of background noise. Existing SHG probes are constructed from heavy metals and are cellular exogenous, presenting with high cytotoxicity, difficult cellular uptake, and the limitation of non-heritability. We, therefore, initially propose an innovative gene-encoded bioprotein SHG probe derived from Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin. The primitive gene of AcMNPV polyhedrin was codon-optimized and mutated in its nuclear localization sequence to achieve cytoplasmic expression in mammalian cells. While providing strong SHG signals, this gene-modified AcMNPV (GM-AcMNPV) polyhedrin could be utilized as an SHG probe for cell imaging. Our experimental results demonstrated successful expression of GM-AcMNPV polyhedrin in the cytoplasm of HEK293T cells and bone mesenchymal stem cells (BMSCs), and verified its characteristic features as an SHG probe. Such SHG probes exhibit high biocompatibility and showed no hindering of central physiological activities such as the differentiation of stem cells. Most importantly, our SHG probes may be successfully used for imaging in living cells. This work will inspire the development of gene encoding-derived bioprotein SHG probes, for long-term tracing of cells/stem cells along with their division, to understand stem cell cycles, reveal stem cell-based therapy mechanisms in regenerative medicine, and unravel cell lineage origins and fates in developmental biology, among other potential applications.

The European Biophysical Societies' Association (EBSA) is an association of 32 biophysical societies in Europe dedicated to the promotion of excellence in biophysics. Through cooperation and collaborative activities, EBSA makes a major and positive impact on the European and International biophysics community. Biennial congresses at various European locations, organized by host societies, are a major activity that engages biophysicists with the wider international scientific community. The European Biophysics Journal, EBJ, is owned by EBSA and publishes high-quality biophysics contributions from around the Globe. The inception of EBSA can be dated to 1984. Peter Bayley, President of EBSA 1990-1993 and Managing Editor, European Biophysics Journal 1984-1999, wrote a history of 'EBSA- the early days', which was published in the Abstract book of the 2007 EBSA Congress. In the present article we aim to update and expand the history to 2024, the 40th anniversary of EBSA, highlighting some developments and achievements of EBSA and the communities it represents.

Human Snk is an evolutionarily conserved serine/threonine kinase essential for the maintenance of endocrine stability. The protein consists of a N-terminal catalytic domain and a C-terminal polo-box domain (PBD) that determines subcellular localization and substrate specificity. Here, an integrated strategy is described to explore the vast structural diversity space of Snk PBD-binding phosphopeptides at a molecular level using machine learning modeling, annealing optimization, dynamics simulation, and energetics rescoring, focusing on the recognition specificity and motif preference of the Snk PBD domain. We further performed a systematic rational design of potent phosphopeptide ligands for the domain based on the harvested knowledge, from which a few potent binders were also confirmed by fluorescence-based assays. A phosphopeptide PP17 was designed as a good binder with affinity improvement by 6.7-fold relative to the control PP0, while the other three designed phosphopeptides PP7, PP13, and PP15 exhibit a comparable potency with PP0. In addition, a basic recognition motif that divides potent Snk PBD-binding sequences into four residue blocks was defined, namely [Χ_-5Χ-₄]_block1-[Ω_-3Ω_-2Ω_-1]_block2-[pS₀/pT₀]_block3-[Ψ₊₁]_block4, where the X represents any amino acid, Ω indicates polar amino acid, Ψ denotes hydrophobic amino acid, and pS₀/pT₀ is the anchor phosphoserine/phosphothreonine at reference residue position 0.