Core-shell model of the clusters of CPEB4 isoforms preceding liquid-liquid phase separation.

Protein solutions can undergo liquid-liquid phase separation (LLPS), where a dispersed phase with a low protein concentration coexists with coacervates with a high protein concentration. We focus on the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein, CPEB4NTD, and its isoform depleted of the Exon4, CPEB4Δ4NTD. They both exhibit LLPS, but in contrast to most systems undergoing LLPS, the single-phase regime preceding LLPS consists mainly of soluble protein clusters. We combine experimental and theoretical approaches to resolve the internal structure of the clusters and the basis for their formation. Dynamic light scattering and atomic force microscopy show that both isoforms exhibit clusters with diameters ranging from 35 to 80 nm. Electron paramagnetic resonance spectroscopy of spin-labeled CPEB4NTD and CPEB4Δ4NTD revealed that these proteins have two distinct dynamical properties in both the clusters and coacervates. Based on the experimental results, we propose a core-shell structure for the clusters, which is supported by the agreement of the dynamic light scattering data on cluster size distribution with a statistical model developed to describe the structure of clusters. This model treats clusters as swollen micelles (microemulsions) where the core and the shell regions comprise different protein conformations, in agreement with the electron paramagnetic resonance detection of two protein populations. The effects of ionic strength and the addition of 1,6-hexanediol were used to probe the interactions responsible for cluster formation. While both CPEB4NTD and CPEB4Δ4NTD showed phase separation with increasing temperature and formed clusters, differences were found in the properties of the clusters and the coacervates. The data also suggested that the coacervates may consist of aggregates of clusters.

Ebola Virus Glycoprotein Strongly Binds to Membranes in the Absence of Receptor Engagement.

Ebola virus (EBOV) is an enveloped virus that must fuse with the host cell membrane in order to release its genome and initiate infection. This process requires the action of the EBOV envelope glycoprotein (GP), encoded by the virus, which resides in the viral envelope and consists of a receptor binding subunit, GP1, and a membrane fusion subunit, GP2. Despite extensive research, a mechanistic understanding of the viral fusion process is incomplete. To investigate GP-membrane association, a key step in the fusion process, we used two approaches: high-throughput measurements of single-particle diffusion and single-molecule measurements with optical tweezers. Using these methods, we show that the presence of the endosomal Niemann-Pick C1 (NPC1) receptor is not required for primed GP-membrane binding. In addition, we demonstrate this binding is very strong, likely attributed to the interaction between the GP fusion loop and the membrane's hydrophobic core. Our results also align with previously reported findings, emphasizing the significance of acidic pH in the protein-membrane interaction. Beyond Ebola virus research, our approach provides a powerful toolkit for studying other protein-membrane interactions, opening new avenues for a better understanding of protein-mediated membrane fusion events.