Biophysical reports最新文献

In adult cardiomyocytes, the type 2a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a vital role in intracellular Ca²⁺ regulation. Reduced SERCA2a function has been associated with decreased myocardial contraction and cardiac output in several heart diseases. Consequently, increasing SERCA2a activity is a high-priority target for treating cardiac pathologies associated with abnormal Ca²⁺ homeostasis. In our previous SERCA ATPase-based screening study, we identified several small molecules as potential activators of SERCA2a function, including the Compound 9, a piperidinyl amide. With porcine cardiac sarcoplasmic reticulum (SR) preparations, we confirmed activation of both SERCA2a ATPase and Ca²⁺-uptake activities. In the current study, we analyzed the effect of Compound 9 on SERCA2a activity on intracellular Ca²⁺ dynamics in ventricular myocytes. Using FRET with human SERCA2a overexpressed in mammalian cells, we confirm that Compound 9 binds and alters SERCA structural dynamics independent of peptide regulators, including phospholamban (PLB). Confocal microscopy and in-cell Ca²⁺ imaging revealed that Compound 9 enhanced Ca²⁺ dynamics in mouse ventricular myocytes. Compound 9 (10 μM) increased the action potential- induced Ca²⁺ transients by 65% and SR Ca²⁺ load by 29%. Moreover, Compound 9 increased Ca²⁺ dynamics during adrenergic receptor stimulation and in PLB knock-out cardiomyocytes, suggesting the stimulatory effect of Compound 9 is PLB independent. Overall, Compound 9 displays characteristics that can be beneficial to enhance cardiac intracellular Ca²⁺ dynamics by increasing SERCA2a function.

E-hooks, or the C-terminal tails of tubulin, mediate interactions between microtubules and associated proteins. Despite their functional importance in cellular and physiological processes, their structural variability and mechanistic roles remain poorly understood. E-hooks are thought to be intrinsically disordered to some degree, making crystallographic studies difficult and necessitating the use of computational tools to study their structures and how they change E-hook function. This review synthesizes recent computational efforts to elucidate E-hook structure, dynamics, and functional differentiation across tubulin isotypes. We examine studies probing E-hooks in isolation or with globular cores, which have revealed subunit-specific features influencing microtubule behavior. We also evaluate the role of E-hooks in modulating binding affinity and conformational states of motor proteins and microtubule-associated proteins (MAPs). Finally, we highlight adjacent technological and methodological advances that have implications for both the interpretation of past findings and the design of future studies, offering new directions for the investigation of E-hook-mediated microtubule regulation.

Hemostasis prevents bleeding by forming clots, though disruptions can cause insufficient or excessive clotting. Shear stress is a critical factor influencing the normal function of the blood and can stimulate the formation of unnecessary clots. Stenosed vessels significantly contribute to this phenomenon by increasing shear stress on the vessel walls. This study aims to quantify the effects of shear stress and stenosis severity on thrombus formation in patient-specific left coronary bifurcations. Three-dimensional patient-specific geometries were reconstructed from angiographic data. Blood flow was modeled using the Brinkman equation, while transport and interactions of coagulation factors were simulated through the convection-diffusion-reaction equation. Model predictions were validated using literature data. The findings showed that all patients experienced significant shear stress at the stenosed regions, which are highly susceptible to clot formation. Shear stress was found to be inversely related to vessel diameter and directly related to the stenosis degree. Furthermore, in bifurcated vessels, blood flow may reach zero prior to complete occlusion by the clot, emphasizing the role of hydrodynamic resistance in redirecting blood flow. Among these factors, the stenosis degree emerges as the most significant predictor of blood flow cessation time. A relationship was developed allowing physicians to accurately and rapidly monitor a patient's condition using angiographic images, providing new insights into the hemodynamic mechanisms of coronary thrombosis and supporting improved diagnosis and treatment of cardiovascular diseases.

Fungi and bacteria are found living in a wide variety of environments, and their interactions are important in many processes including soil health, human and animal physiology, and in biotechnological applications. Here, we investigate a single morphological feature of cocultures of planktonic bacterial growth within biofilm-forming liquid cultures of mycelium, namely, the attachment of bacterial ectobionts of species Bacillus subtilis to fungal hyphae of species Hericium erinaceus. The bacteria-in-mycelial-biofilm method was developed and utilized to allow for attachment of bacteria to hyphae via containment within exopolymeric substances (EPS) and the overall extracellular matrix of the mycelium. A graded dehydration protocol was used to selectively remove extraneous biofilm components and reveal intact bacteria and surface-interfacing features. The dehydration methods allowed for identification of specific interactions and differentiated these cultures from trivial stochastic mixing of bacteria and mycelium in liquid media. Attachment structures appear to be produced primarily by the mycelium and enveloped the bacterial ectobiont. Nanoscale surface-interfacing EPS constituents were preserved, providing a biophysical basis for a range of contact-dependent modulating properties of the bacteria on this fungal host. The mean biofilm area across triplicates was 3.90μm²±0.72μm², and the mean percentage coverage was 18.33%±5.52%. The bacterial biofilm components could not be ruled out as co-contributing to formation of attachment structures due to the structures being present connecting individual bacteria as well as to hyphae.

The degree of over-/underwinding of the DNA double helix, quantified by the superhelical density, is a key feature modulating critical biological processes such as gene expression and regulation. In fact, DNA molecules are able to channel the excess levels of mechanical stress into local defective and denatured states that are promptly detected by, e.g., transcription factors and nuclease enzymes. The occurrence and stability of these motifs are dictated by a complex interplay between topological and sequence-dependent effects, ultimately affecting the global conformational dynamics of the DNA molecule itself. Here, we characterize the impact of the sequence and of the superhelical density on the structural evolution of a 672-bp DNA minicircle via classical molecular dynamics simulations employing the coarse-grained oxDNA force field. We observe that moderately-to-highly undercoiled regimes are associated with the occurrence of stable, somewhat broad denaturation bubbles, typically co-localizing with flexible nucleotide sequences on the DNA minicircle. These defects are hardly reabsorbed by the system, thereby pinning the subsequent dynamics of the molecule. In fact, a similar behavior was recapitulated by enforcing "synthetic,"adjoining DNA mismatches, regardless of the underlying nucleotide sequence, suggesting an effective manner of DNA manipulation.

Crystallins are highly stable, soluble proteins that refract light and maintain transparency in the vertebrate eye lens. They are not replaced after early development, making them an excellent system for studying protein stability and solubility in crowded environments. To better understand the effects of deamidation on these ubiquitous vertebrate crystallins, we investigated a particularly extreme example, a lens protein from the long-lived Antarctic toothfish (Dissostichus mawsoni), γS1 crystallin (DmγS1). This protein remains soluble in the crowded fish lens, maintaining its transparency even at -2°C and at concentrations more than twofold that of humans (nearly 1000 mg/mL) and over a comparable timescale. As the organism ages, crystallins accumulate oxidative damage such as deamidation of Asn and Gln side chains, leading to aggregation and cataract. Previous studies of human γS crystallin (HγS) have shown that extensive deamidation reduces stability and increases aggregation propensity. Here, we present the biophysical characterization of wild-type DmγS1 and variants with three, five, and seven deamidation sites. In sharp contrast to results for human γS-crystallin, increasing the number of deamidations does not significantly change the thermal stability of DmγS1. These proteins are startlingly resistant to thermal denaturation; despite their psychrophilic origin, they have midpoint unfolding temperatures between 56°C and 63°C. Extensive deamidation does make the protein more vulnerable to chemical denaturation as well as aggregation below the unfolding temperature; however, all the variants resist aggregation well above the fish's physiological temperature. These proteins present a useful model system for aggregation resistance in extreme environments; most studies of protein solubility focus on unusually aggregation-prone proteins, but understanding the underlying biophysics also requires studying extremely soluble proteins.

Measuring dipole orientations of fluorescent probes offers unique local structural insights of labeled biomolecules and has seen expanding applications in structural biology studies. Here, we propose an alternative imaging geometry, "dual-view" microscopy, for single-molecule dipole orientation measurements. We develop a protocol capable of simultaneously measuring absorption and emission dipole orientations of single emitters. Further, through simulation, we demonstrate that absorption dipole orientation can be accurately measured with high and uniform precision in three dimensions, significantly outperforming epifluorescence microscopy. Meanwhile the emission dipole is independently narrowed down to four possible orientations and can be uniquely determined with the co-estimated absorption dipole. Dual-view microscopy represents a new paradigm in single-molecule orientation sensing and could have applications in imaging under cryogenic temperatures.

Protein condensation is the basis for formation of membrane-less organelles in the cell. Most famously, weak, polyvalent interactions, often including RNA, may lead to a liquid-liquid phase separation. This effect greatly enhances local concentrations and is thought to promote interactions that would remain rare in dilute solution. Synthetic systems provide a means to clarify the underlying biophysical mechanisms at play, both in vitro and in the cell via exogenous expression. In this regard, ferritin is a useful substrate, as its composition of 24 subunits with octahedral symmetry supports self-assembly by close packing in 3D. The conventional diagnostic tool for protein condensation is fluorescence imaging. In this work, we explore the use of refractive index mapping to detect states of condensation and decondensation. Using two related ferritin-based self-assembly systems, we find that refractive index is a sensitive indicator for reversible condensation. Surprisingly, refractive index indicates a rapid decondensation even when molecular dispersal kinetics are slow according to fluorescence. Conversely, in a photoactivated condensation where long activation results in slow decondensation kinetics, the refractive index provides reliable evidence for the physical state independent of fluorescence. The observations suggest a distinction between condensation to a sparse biomolecular network or to a material continuum that supports an optical polarizability distinct from that of the dilute phase in solution.

The presented study aimed to investigate the antibacterial activity of menthol-the main component of one of the widespread plants of the Lamiaceae family-Mentha arvensis. To investigate the mode of action of menthol, we studied its influence on kanamycin-resistant E. coli pARG-25 and wild-type E. coli BW25113 strains. For this, the effect of menthol on ATPase activity, proton and potassium fluxes, and intracellular pH was investigated under aerobic and anaerobic conditions. The results showed that menthol influences these parameters in a concentration- and condition-dependent way. It likely interacts with FoF₁-ATPase and other systems involved in energy-generating processes and ion transport, disrupting the bacterial metabolism of both antibiotic-resistant and -susceptible strains.

Skeletal muscle alpha actin (ACTA1) is important for muscle contraction and relaxation, with historical studies focused on ACTA1 mutations in muscle dysfunction. Proteomics reports have consistently observed that actin, including ACTA1, is acetylated at multiple lysine sites. However, few reports have studied the effects of actin acetylation on cellular function, and fewer have examined ACTA1 acetylation on skeletal muscle function. Here, we aimed to examine how ACTA1 acetylation affected actomyosin interactions by determining actin sliding velocity, myosin binding, and calcium sensitivity. In this study, ACTA1 was chemically acetylated via acetic anhydride (AA) to increasing levels of acetylation: low-level acetylation (using 0.1 mM AA), mid-level acetylation (0.3 mM AA), and high-level acetylation (1 mM AA). We report that ACTA1 acetylation significantly decreased actin sliding velocity and actin filament length. Further analysis showed that ACTA1 acetylation significantly increased calcium sensitivity, with a loss of tropomyosin regulation noted with high-level ACTA1 acetylation. Lastly, ACTA1 acetylation enhanced skeletal myosin half maximal binding to actin. These data highlight acetylation as an additional posttranslational modification, outside of phosphorylation, in the regulation of muscle contraction and skeletal muscle alpha actin function.