Biophysical journal最新文献

The dynamics of many macromolecular machines is characterized by chemically-mediated structural changes that achieve large scale functional deployment through local rearrangements of constitutive protein sub-units. Motivated by recent high resolution structural microscopy of a particular class of such machines, contractile injection systems (CIS), we construct a coarse grained semi-analytical model that recapitulates the geometry and bistable mechanics of CIS in terms of a minimal set of measurable physical parameters. We use this model to predict the size, shape and speed of a dynamical actuation front that underlies contraction. Scaling laws for the velocity and physical extension of the contraction front are consistent with our numerical simulations, and may be generally applicable to related systems.

Supraphysiological shear rates (>2,000 s^-1) amplify von Willebrand factor (vWF) unfurling and increase platelet activation and adhesion. These elevated shear rates and shear rate gradients also play a role in shear-induced platelet aggregation (SIPA). The primary objective of this study is to investigate the contributions of major binding receptors to platelet deposition and SIPA in a stenotic model. Microfluidic channels with stenotic contractions ranging from 0-75% are fabricated and coated with human type I collagen at 100 μg/mL. Fresh human blood is reconstituted to 40% HCT and treated to stain platelets. Platelet receptors α_IIbβ₃, GPIb, or GPVI are blocked with inhibitory antibodies or proteins to reduce platelet function under flow at 500, 1,000, 5,000, or 10,000 s^-1 over 5 minutes of perfusion. Additional validation experiments are performed by dual-blocking receptors and performing coagulability testing by rotational thromboelastometry. Control samples exhibit SIPA correlating to increasing shear rate and increasing stenotic contraction. Inhibition of α_IIbβ₃ or GPIb receptors causes a nearly total reduction in platelet adhesion and a loss of aggregation at >1,000 s^-1. GPVI inhibition does not notably reduce platelet adhesion at 500 or 1,000 s^-1 but affects microthrombus stability at 5-10,000 s^-1 following aggregation formation in 50-75% stenotic channels. Inhibition of vWF-binding receptors completely blocks adhesion and aggregation at shear rates >1,000 s^-1. Inhibition of GPVI reduces platelet adhesion at 5-10,000 s^-1 but renders thrombi susceptible to fragmentation. This study yields further insight into mechanisms regulating rapid growth and stabilization of arterial thrombi at supraphysiological shear rates.

Cells can dynamically organize reactions through the formation of biomolecular condensates. These viscoelastic networks exhibit complex material properties and mesoscale architectures, including the ability to form multi-phase assemblies. It was shown previously that condensates with complex architectures may arise at equilibrium in multicomponent systems or in condensates that were driven out-of-equilibrium by changes in external parameters such as temperature. In this study, we demonstrate that the aging of initially homogeneous protein-RNA condensates can spontaneously lead to the formation of kinetically arrested double-emulsion and core-shell structures without changes in external variables such as temperature or solution conditions. By combining time-resolved fluorescence-based experimental techniques with simulations based on the Cahn-Hilliard theory, we show that, as the protein-RNA condensates age, the decrease of the relative strength of protein-RNA interactions induces the release of RNA molecules from the dense phase. In condensates exceeding a critical size, aging combined with slow diffusion of the macromolecules trigger nucleation of dilute phase inside the condensates, which leads to the formation of double-emulsion structures. These findings illustrate a new mechanism of formation of multi-compartment condensates.

Synaptotagmin-1 (syt1) functions as the Ca²⁺-dependent sensor that triggers the rapid and synchronous release of neurotransmitters from neurotransmitter-containing vesicles during neuronal exocytosis. The syt1 protein has two homologous tandem C2 domains that interact with phospholipids in a Ca²⁺-dependent manner. Despite the crucial role of syt1 in exocytosis, the precise interactions between Ca²⁺, syt1, and phospholipids are not fully understood. In a study involving recessive lethal mutations in the syt1 gene, a specific mutation named AD3 was generated in Drosophila syt1, resulting in a significant reduction in Ca²⁺-dependent exocytosis. Further investigation revealed that the AD3 mutation was a missense mutation located in a conserved consensus sequence within the C2B domain of Drosophila syt1. However, the biophysical impact of the AD3 mutation had not been analyzed. Our study uses X-ray crystallography, isothermal titration calorimetry (ITC), thermodynamic analysis, and molecular dynamics simulation to show that the primary defect caused by the AD3 mutation in the syt1 protein is reduced thermodynamic stability. This instability alters the population of Ca²⁺-receptive states, leading to two major consequences: decreased affinity for calcium ions and compromised stabilization of the domain normally enhanced by Ca²⁺. We conclude that this conserved residue acts as a structural constraint, delimiting the movement of loop 3 within the pocket and ultimately influencing the affinity of calcium ion binding with the C2 domain.

The flaviviral NS2B/NS3 protease is a conserved enzyme required for flavivirus replication. Its highly dynamic conformation poses major challenges but also offers opportunities for antiviral inhibition. Here, we established a nanopore tweezers-based platform to monitor NS2B/NS3 conformational dynamics in real-time. Molecular simulations coupled with electrophysiology revealed that the protease could be captured in the middle of the ClyA nanopore lumen, stabilized mainly by dynamic electrostatic interactions. We designed a new Salmonella typhi ClyA nanopore with enhanced nanopore/protease interaction that can resolve the open and closed states at the single-molecule level for the first time. We demonstrated that the tailored ClyA could track the conformational transitions of the West Nile NS2B/NS3 protease and unravel the conformational energy landscape of various protease constructs through population and kinetic analysis. The new ClyA-protease platform paves a way to search for new allosteric inhibitors that target the NS2B and NS3 interface.

Influenza A virus particles assemble at the plasma membrane of infected cells. During assembly all components of the virus come together in a coordinated manner to deform the membrane into a protrusion eventually forming a new, membrane-enveloped virus. Here we integrate recent molecular insights of this process, particularly concerning the structure of the matrix protein 1 (M1), within a theoretical framework describing the mechanics of virus assembly. Our model describes M1 polymerization and membrane protrusion formation, explaining why it is efficient for M1 to form long strands assembling into helices in filamentous virions. Eventually, we find how the architecture of M1 helices is controlled by physical properties of viral proteins and the host cell membrane. Finally, by considering the growth force and speed of viral filaments, we propose that the helical geometry of M1 strands might have evolved to optimize for fast and efficient virus assembly and growth.

Weak protein interactions are associated with a broad array of biological functions and are often implicated in molecular dysfunction accompanying human disease. In addition, these interactions are a critical determinant in the effective manufacturing, stability, and administration of biotherapeutic proteins. Despite their prominence, much remains unknown about how molecular attributes influence the hydrodynamic and thermodynamic contributions to the overall interaction mechanism. To systematically probe these contributions, we have evaluated self-interaction in a diverse set of proteins that demonstrate a broad range of behaviors from attractive to repulsive. Analysis of the composite trending in the data provides a convenient interconversion among interaction parameters measured from the concentration dependence of the molecular weight, diffusion coefficient, and sedimentation coefficient, as well as insight into the relationship between thermodynamic and hydrodynamic interactions. We find relatively good agreement between our data and a model for interacting hard spheres in the range of weak self-association. In addition, we propose an empirically derived, general scaling relationship applicable across a broad range of self-association and repulsive behaviors.

DEAD-box helicases use ATP to unwind short double-stranded RNA (dsRNA). The helicase core consists of two discrete domains, termed RecA_N and RecA_C. The nucleotide binding site is harbored in RecA_N, while both RecA_N and RecA_C are involved in RNA recognition and ATP hydrolysis. In the absence of nucleotides or RNA, RecA_N and RecA_C do not interact ("open" form of the enzyme). In the presence of both RNA and ATP the two domains come together ("closed" form), building the composite RNA binding site and stimulating ATP hydrolysis. Because of the different roles and thermodynamic properties of the ADP-bound and ATP-bound states in the catalytic cycle, the conformations of DEAD-box helicases in complex with ATP and ADP are assumed to be different. However, the available crystal structures do not recapitulate these supposed differences and show identical conformations of DEAD-box helicases independent of the identity of the bound nucleotide. Here, we use NMR to demonstrate that the conformations of the ATP- and ADP-bound forms of the DEAD-box helicase Vasa are indeed different, contrary to the results from x-ray crystallography. These differences do not relate to the populations of the open and closed forms, but are intrinsic to the RecA_N domain. NMR chemical shift analysis reveals the regions of RecA_N where the average conformations of Vasa-ADP and Vasa-ATP are most different and indicates that these differences may contribute to modulating the affinity of the two nucleotide-bound complexes for RNA substrates.