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Glycerophospholipids are synthesized primarily in the cytosolic leaflet of the endoplasmic reticulum (ER) membrane and must be equilibrated between bilayer leaflets to allow the ER and membranes derived from it to grow. Lipid equilibration is facilitated by integral membrane proteins called "scramblases." These proteins feature a hydrophilic groove allowing the polar heads of lipids to traverse the hydrophobic membrane interior, similar to a credit card moving through a reader. Nevertheless, despite their fundamental role in membrane expansion and dynamics, the identity of most scramblases has remained elusive. Here, combining biochemical reconstitution and molecular dynamics simulations, we show that lipid scrambling is a general feature of protein insertases, integral membrane proteins which insert polypeptide chains into membranes of the ER and organelles disconnected from vesicle trafficking. Our data indicate that lipid scrambling occurs in the same hydrophilic channel through which protein insertion takes place and that scrambling is abolished in the presence of nascent polypeptide chains. We propose that protein insertases could have a so-far-overlooked role in membrane dynamics as scramblases. [ABSTRACT FROM AUTHOR]

The ionizable-lipid component of RNA-containing nanoparticles controls the pH-dependent behavior necessary for an efficient delivery of the cargo—the so-called endosomal escape. However, it is still an empirical exercise to identify optimally performing lipids. Here, we study two well-known ionizable lipids, DLin-MC3-DMA and DLin-DMA using a combination of experiments, multiscale computer simulations, and electrostatic theory. All-atom molecular dynamics simulations, and experimentally measured polar headgroup pKa values, are used to develop a coarse-grained representation of the lipids, which enables the investigation of the pH-dependent behavior of lipid nanoparticles (LNPs) through Monte Carlo simulations, in the absence and presence of RNA molecules. Our results show that the charge state of the lipids is determined by the interplay between lipid shape and headgroup chemistry, providing an explanation for the similar pH-dependent ionization state observed for lipids with headgroup pKa values about one-pH-unit apart. The pH dependence of lipid ionization is significantly influenced by the presence of RNA, whereby charge neutrality is achieved by imparting a finite and constant charge per lipid at intermediate pH values. The simulation results are experimentally supported by measurements of α-carbon 13C-NMR chemical shifts for eGFP mRNA LNPs of both DLin-MC3-DMA and DLin-DMA at various pH conditions. Further, we evaluate the applicability of a mean-field Poisson–Boltzmann theory to capture these phenomena. [ABSTRACT FROM AUTHOR]