guanosine-triphosphate and 1-palmitoyl-2-oleoylphosphatidylcholine

Cell division is the most dynamic event in the cell cycle. Recently, efforts have been made to reconstruct it using the individual component proteins to obtain a better understanding of the process of self-reproduction of cells. However, such reconstruction studies are frequently hampered by difficulties in preparing membrane-associated proteins. Here we demonstrate a de novo synthesis approach based on a cell-free translation system. Genes for fundamental cell division proteins, FtsZ, FtsA, and ZipA, were expressed inside the lipid compartment of giant vesicles (GVs). The synthesized proteins showed polymerization, membrane localization, and eventually membrane deformation. Notably, we found that this morphological change of the vesicle is forced by only FtsZ and ZipA, which form clusters on the membrane at the vesicle interior. Our cell-free approach provides a platform for studying protein dynamics associated with lipid membrane and paves the way to create a synthetic cell that undergoes self-reproduction.

Topics: Bacterial Proteins; Carrier Proteins; Cell Cycle Proteins; Cell Membrane; Cell-Free System; Cytoskeletal Proteins; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Green Fluorescent Proteins; Guanosine Triphosphate; Hydrolysis; Imaging, Three-Dimensional; Phosphatidylcholines; Phosphatidylglycerols; Protein Engineering; Unilamellar Liposomes

Heterotrimeric G-proteins' activation on the intracellular side of the cell membrane is initiated by stimulation of the G-Protein Coupled Receptors (GPCRs) extra-cellular part. This two-step activation mechanism includes (1) an exchange between GDP and GTP molecules in the G(α) subunit and (2) a dissociation of the whole G(αβγ) complex into two membrane-anchored blocks, namely the isolated G(α) and G(βγ) subunits. Although X-ray data are available for both inactive G(αβγ):GDP and active G(α):GTP complexes, intermediate steps involved in the molecular mechanism of the dissociation have not yet been addressed at the molecular level. In this study, we first built a membrane-anchored intermediate G(iαβγ):GTP complex. This model was then equilibrated by molecular dynamics simulations before the Targeted Molecular Dynamics (TMD) technique was used to force the G(α) subunit to evolve from its inactive (GDP-bound) to its active (GTP-bound) conformations, as described by available X-ray data. The TMD constraint was applied only to the G(α) subunit so that the resulting global rearrangements acting on the whole G(αβγ):GTP heterotrimer could be analyzed. We showed how these mainly local conformational changes of G(α) could initiate large domain:domain motions of the whole complex, the G(βγ) behaving as an almost quasi-rigid block. This separation of the two G(α):GTP and G(βγ) subunits required the loss of several interactions at the G(α):G(βγ) interface that were reported. This study provided an atomistic view of the crucial intermediate step of the G-proteins activation, e.g., the dissociation, that could hardly be elucidated by the experiment.

Topics: Crystallography, X-Ray; Guanosine Diphosphate; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Kinetics; Molecular Dynamics Simulation; Phosphatidylcholines; Protein Binding; Protein Subunits; Thermodynamics