European Biophysics Journal最新文献

Enhancing protein stability while maintaining activity is a long-standing challenge in protein engineering, as modifications that benefit one property often compromise another. In this study, we leveraged a computational design strategy, CamSol Combination, to make a first step to improve the stability of purple acid phosphatase (PAP), a metalloprotein known for its distinctive pink color. PAP serves as a challenging model for engineering due to its complex redox-active site and the incorporation of iron ions critical to its function. Five mutations were introduced-H22R, A24P, F54P, H197P, and T208R-targeted to enhance thermal stability, as suggested by the computational design pipeline, while avoiding key functional regions. Experimental validation confirmed the choice of mutations with a 5 °C increase in thermal stability and retained enzymatic activity across a slightly expanded pH range. The mutations introduced subtle shifts in the enzyme's spectral and redox behavior, consistent with a lower energy of the oxidized state, and with dynamic light scattering data suggesting low aggregation. These results highlight the potential of computational approaches like the CamSol Combination to streamline protein engineering by enabling multi-trait optimization.

Curcumin (CUR), a bioactive compound extracted from the turmeric (Curcuma longa), has gathered considerable attention in recent years due to its claimed health benefits, including anti-inflammatory, antioxidant, and neuroprotective properties. The dysregulation of ion channel activity and the altered neuronal excitability in neurons has been identified as a key factor in the pathophysiology of neurological disease and a putative pharmacological target for therapeutic options. Therefore, we investigated by whole-cell patch-clamp the CUR's impact on the ionic currents in motoneuron-derived (MN-1) cells modeling SBMA and in human neuro-progenitor-cell (hNPCs)-derived neurons. CUR decreased viability in non-pathological MN-1 cells but showed increased resistance in pathological MN-1 cells, while mature neurons derived from hiPSCs remained unaffected under the same conditions. Electrophysiological studies revealed that CUR inhibits outward and inward currents in both MN-1 cell types, with a more pronounced effect in pathological cells. In hNPC-derived neurons, CUR also inhibited both currents and induced a negative shift in the voltage dependence of activation, suggesting reduced excitability. Our results indicate that further investigations are needed to confirm the role of CUR in the context of neurotherapeutics based on ion channel-targeting pharmacology.

Vitamin B12 (cobalamin, Cbl) is a coordination compound of the cobalt, located at the center of a corrin ring composed of four pyrrolic-like groups. The cobalt ion can be bound to a variety of upper axial ligands, which vary among different cobalamin forms, including hydroxocobalamin (OHCbl), cyanocobalamin (CNCbl), methylcobalamin (MeCbl), and adenosylcobalamin (AdoCbl). MeCbl and AdoCbl are considered the biologically active forms, serving as cofactors in the metabolism of methylmalonic acid (MMA) and homocysteine (HCY). Impaired conversion of these metabolites leads to their pathological accumulation, resulting in severe cellular damage. This is precisely what occurs in cblC deficiency, a rare inborn disorder caused by mutations in the MMACHC protein, which plays a crucial role in binding and processing the various cobalamin forms. Mutations affecting MMACHC function impair its ability to correctly handle cobalamins, leading to the disease. In this study, we evaluated the impact of various cobalamin forms, specifically AdoCbl, MeCbl, and CNCbl, on the stability and oligomeric organization of the wild type MMACHC protein, using circular dichroism spectroscopy, native gel electrophoresis, and small-angle X-ray scattering. Moreover, isothermal titration calorimetry experiments provided insights into the thermodynamic parameters governing MMACHC binding to these cobalamins. In addition, we also assessed how the R161Q mutation in MMACHC alters the affinity of this protein for the different vitamin B12 forms, leading to decreased stability and impaired homodimerization, a process likely relevant to its functional role. Our findings provide molecular insights into cblC pathogenesis and advance our understanding of MMACHC structure-function relationships.

Biological membranes are highly dynamic and adaptive interfaces that define cellular compartments, posing significant challenges for detailed characterization. Among the diverse range of experimental and computational techniques, small-angle scattering emerges as a label-free, non-invasive method capable of probing membrane structures across length scales from micrometers to subnanometers. By exploiting the complementary contrasts of X-ray and neutron scattering, combined with advanced optimization algorithms, this approach has provided unique insights into membranes with well-defined lipid and protein architectures. In this review, we highlight recent studies from the Pabst Lab, including investigations of lipid domains, asymmetric lipid membranes, and intrinsic lipid curvature. Furthermore, we explore the functional implications of these findings, such as the activity of an integral membrane enzyme and the effects of antimicrobial peptides in live cells. These examples underscore the versatility of small-angle scattering techniques in elucidating membrane functions, offering valuable perspectives for understanding cellular processes and advancing pharmaceutical applications.

We have collected fluorescence images of fixed cell nuclei of two different types-HeLa and HepG2-with DNA labeled by a standard fluorophore, and have devised three different quantitative parameters aimed to describe the distribution of the nuclear chromatin. The parameters are the fractal dimension, associated with the intricacy and hierarchical structure of chromatin; the total perimeter of local maxima, associated with the amount of chromatin domains; and the radial distance of angularly averaged intensity profile maximum, associated with the possible occurrence of a peak density at a characteristic distance from the nucleus center. Our results suggested that it was possible to differentiate the two types of cells in the 3D space of the defined parameters. Therefore, these parameters appear promising in identifying specific functional patterns in chromatin. At the same time, the negative control of different runs of measurements on the same cell type also showed at least partial differentiation. Thus, the tool proposed here for nuclear chromatin pattern characterization is probably sensitive to the cell life cycle moment almost as much as to the cell type and should be tested further on cells synchronized at the same phase during their cycle.