European Biophysics Journal最新文献

The stability of membrane proteins depends on amino acid-specific interactions within the protein structure, its surroundings, e.g. the lipid membrane and ionic solutions, and their substrates. A molecular understanding of the factors affecting protein stability is essential for comprehending the respective structure-function relationships. This study aims to investigate the influence of native cysteine residues, ions, and glycerol on the stability of GlpF, the aqua(glycerol)porin of E. coli, under well-controlled conditions. To this end, the wild-type protein and its variant with four cysteine residues in the transmembrane helix bundle mutated to glycines were overexpressed and purified. The thermal unfolding of GlpF occurs at a transition temperature of 59.2 ± 1.4 °C, corresponding to an apparent Gibbs free energy of unfolding of approximately 10 kcal/mol. In contrast to the wild-type protein, the mutated GlpF exhibits a second unfolding transition, lowered by approximately 10 °C, and a decreased Gibbs free energy of unfolding of 5 kcal/mol. Our findings highlight that divalent ions and glycerol have a more pronounced stabilizing effect on the mutated GlpF than the wild-type protein. The structural consequences of cysteine to glycine mutations in terms of transmembrane helix rearrangement, the inter- and intra-chain H-bond network, and the reduced inter-chain interaction free energy, as well as intra-chain electrostatic energy and van der Waals energy, are revealed by molecular dynamics simulations. In conclusion, our findings illustrate the significance of cysteine residues distant from each other on protein oligomerization and stability, and the amplified impact of (de)stabilizing agents on less stable protein variants.

The ordered mesoporous silica SBA-15 is a promising platform for protein immobilization and delivery, since adsorption into its porous matrix may improve protein structural stability. Here, three proteins-myoglobin (Mb), concanavalin A (ConA), and soybean trypsin inhibitor (STI)-were loaded into conventional SBA-15 particles with an average mesopore diameter of 9.3 nm to investigate structural changes in their predominant content after adsorption. Powdered biocomposites were prepared by loading each protein at two silica-to-protein weight ratios (10:1 and 5:1). Protein structures were evaluated by synchrotron radiation circular dichroism and fluorescence spectroscopy following resuspension in buffer solutions (pH 7.0 and 2.2). The structural changes were qualitatively analyzed and found to be more influenced by the silica-to-protein ratio for Mb and ConA, while buffer pH had a lesser effect on the proteins. A 10:1 ratio better preserved the α-helical spectral features of Mb, whereas a 5:1 ratio favored the retention of the β-sheet signature of ConA; in both cases, the aromatic residue microenvironment remained largely preserved. In contrast, STI, rich in irregular structures, exhibited smaller spectral changes relative to its native structure in both ratios, while the aromatic residue was less exposed to the solvent. Overall, the results reinforce the potential of SBA-15 as a protein carrier by retaining the main features of the protein's secondary structure under specific conditions. The simultaneous comparison of three structurally distinct proteins reveals variable adsorption behaviors and highlights the silica-to-protein ratio as a protein-dependent parameter, reinforcing the importance of optimizing adsorption conditions to develop more stable silica-protein systems.

The Hfq protein is not only a mediator of RNA metabolism but also a key structural element involved in nucleic acid shaping. Its ability to compact and organize DNA, as well as its influence on the dynamics of various DNA-related processes, makes Hfq a central player in the regulation of bacterial chromosomal architecture and function. We previously demonstrated that different DNA methylation states affect Hfq binding and mobility. In this study, we show that Hfq, through its C-terminal region, can influence a DNA entangled/disentangled transition and examine the impact of DNA methylation on this previously uncharacterized function of Hfq. This discovery provides new insights into the role of Hfq in DNA transactions, with potential implications for essential cellular processes such as recombination and replication. Furthermore, this study demonstrates that Synchrotron Radiation Linear Dichroism (SRLD) is a powerful tool that can follow cooperative vs non-cooperative protein induced DNA structural transitions.