Description
Phage shock protein A (PspA) is a membrane-associated protein that is believed to play a critical role in bacterial membrane fusion, yet, its mechanism is less understood. In this study, we reconstituted the cell-free PspA synthesis within liposomes and observed its phenotypic effects on membranes. This process is highly critical for the development of a self-sustained artificial cell model with cell-like properties induced by self-synthesized proteins that are generated from its genetic level. In this study, we successfully designed multiple plasmids of pspA and translated them to PspA using cell-free protein synthesis (containing in-vitro transcription and translation molecules extracted from E. coli) in both bulk and liposomes. In particular, PspA contains 5 α-helices (α1-α5); and here, the process of synthesis of each truncated α-helix was also successfully demonstrated. Moreover, cell-free synthesis of PspA (full-length, α1, α12, α123, and α1234) in a bulk system revealed aggregation and oligomerization (self-assembly) and formed μm sized filament-like structures, highlighting the critical role of α1 on polymerization and filament formation. Interestingly, when encapsulated within liposomes, these proteins induced the shape change in the liposomal membrane to be more elongated. In Cryo-EM analysis, these proteins are capable of binding with membranes, creating rapture (hole-like structure), and deforming liposomes into several shapes, such as tubule membranes, elongated membranes, internal budding, and endocytosis (fission). This result implies that PspA (mainly through α1) may somehow remodel the membranes through interactions and further induce deformation. Overall, we highlight that α1 plays vital roles in PspA aggregation/polymerization, membrane interaction, rapture, fission, and shape deformation. We assume that these phenotypes may be the intermediate process of membrane fusion.
