European Biophysics Journal最新文献

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.

Uptake of gold nanoparticles by HeLa cells fixed at different incubation times of up to eight hours was investigated using 3D confocal laser scanning microscopy followed by image analysis. The cell bodies were characterized by fluorescence labeling, whereas the gold nanoparticles were identified by light scattering. The amount of nanoparticles uptaken at different times was evaluated with Fiji according to a dedicated protocol. We assessed the effect of particle size (80 and 150 nm diameter spheres) and shape (spheres vs "urchins") on their uptake. The large spherical nanoparticles presented approximately fourfold higher levels of uptake than the nanourchins. Also, the spheres presented a clearly increasing uptake in the time profile, reaching a maximum around seven hours and then showing a slight decrease. We ascribe this behavior to a lower aptitude of the cells for taking up nanoparticles with either urchin shape or smaller size and to the possible presence of competing kinetics for exocytosis at the longest times. Live experiments confirmed for the time profile a relatively flat cell response to the urchins and an increasing response to the spheres, yet with a final plateau. However, in the live case, the internalization levels were as low for the spheres as for the urchins.

The SARS-CoV-2 non-structural protein 1 (Nsp1) acts at multiple points toward the host cell to trigger its mRNA cleavage and decay. Nsp1 is found binding with the 40S ribosomal subunit and inhibiting the translation process, as well as docking with different cyclophilins. Herein, we evaluated the structural physicochemical properties of SARS-CoV-2 Nsp1 protein implementing different computational techniques. The Nsp1 was found to form a structured α-helical C-terminal region, following a conformational switch at residue S166 that is necessary for binding the 40S ribosome subunit. Similarly, the presence of cyclophilins stabilizes the Nsp1 C-terminus making a tilt movement at position 166. In the 40S ribosome-Nsp1 machinery, both the ribosomal uS3 and eS30 components were found equally interacting with Nsp1, which guided construction of their pharmacophores. Among a set of studied cyclophilins, FKBP1B showed the highest affinity with Nsp1 and PPIH made least interactions. The majority of cyclophilins dock to the conserved Nsp1 loop or linker region, which connects the C-terminus to the central domain. Our findings revealed that Nsp1 has a versatile C-terminus region which changes its conformations with respect to its host binding partner. Identified novel binding sites within the Nsp1 can assist in understanding its networking (in current or future such infections), as well as support drug discovery programs aimed at targeting the coronavirus family.