Effect of bacterial cellulose nanofibers incorporation on acid-induced casein gels: microstructures and rheological properties

In this study, the effect of bacterial cellulose nanofibers (BCNF) incorporation on the structural and rheological properties of casein gels was investigated, where the mixed BCNF and casein gels were prepared by adding gluconic acid δ-lactone (GDL) to acidify the mixed polymer solutions at 3.0% casein concentration (w/v) and varying BCNF concentrations (0–0.5%, w/v). By changing the addition amount of GDL, the mechanical and structural properties of the mixed gels were studied at above, near and below the electric point (pI) of the casein. At pH above the pI of the casein, the introduction of BCNF initially increased the gel strength, but further addition of BCNF weakened the mixed gels. At near and below the pI of the casein, the incorporation of BCNF continuously increased the gel strength. Besides, all gels showed good structural homogeneity, without macroscopic phase separation occurring, which indicated good compatibility of BCNF with the casein gels.

Phase Separation of Shell Protein and Enzyme: An Insight into the Biogenesis of a Prokaryotic Metabolosome

Bacterial microcompartments are substrate specific metabolic modules that are conditionally expressed in certain bacterial species. These all protein structures have size in the range of 100-150 nm and are formed by the self-assembly of thousands of protein subunits, all encoded by genes belonging to a single operon. The operon contains genes that encode for both enzymes and shell proteins. The shell proteins self-assemble to form the outer coat of the compartment and enzymes are encapsulated within. A perplexing question in MCP biology is to understand the mechanism which governs the formation of these small yet complex assemblages of proteins. In this work we use 1,2-propanediol utilization microcompartments (PduMCP) as a paradigm to identify the factors that drive the self-assembly of MCP proteins. We find that a major shell protein PduBB tend to self-assemble under macromolecular crowded environment and suitable ionic strength. Microscopic visualization and biophysical studies reveal phase separation to be the principle mechanism behind the self-association of shell protein in the presence of salts and macromolecular crowding. The shell protein PduBB interacts with the enzyme diol-dehydratase PduCDE and co-assemble into phase separated liquid droplets. The co-assembly of PduCDE and PduBB results in the enhancement of catalytic activity of the enzyme. A combination of spectroscopic and biochemical techniques shows the relevance of divalent cation Mg2+ in providing stability to intact PduMCP in vivo. Together our results suggest a combination of protein-protein interactions and phase separation guiding the self-assembly of Pdu shell protein and enzyme in solution phase.

Experimental Evidence of Intrinsic Disorder and Amyloid Formation by the Henipavirus W Proteins

Henipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V and W proteins which share with P their N-terminal, intrinsically disordered domain (NTD) and possess a unique C-terminal domain. Henipavirus W proteins antagonize interferon (IFN) signaling through NTD-mediated binding to STAT1 and STAT4, and prevent type I IFN expression and production of chemokines. Structural and molecular information on Henipavirus W proteins is lacking. By combining various bioinformatic approaches, we herein show that the Henipaviruses W proteins are predicted to be prevalently disordered and yet to contain short order-prone segments. Using limited proteolysis, differential scanning fluorimetry, analytical size exclusion chromatography, far-UV circular dichroism and small-angle X-ray scattering, we experimentally confirmed their overall disordered nature. In addition, using Congo red and Thioflavin T binding assays and negative-staining transmission electron microscopy, we show that the W proteins phase separate to form amyloid-like fibrils. The present study provides an additional example, among the few reported so far, of a viral protein forming amyloid-like fibrils, therefore significantly contributing to enlarge our currently limited knowledge of viral amyloids. In light of the critical role of the Henipavirus W proteins in evading the host innate immune response and of the functional role of phase separation in biology, these studies provide a conceptual asset to further investigate the functional impact of the phase separation abilities of the W proteins.