Constructing Lipid Tools for Biochemical Applications

Synthetic membranes have long been a powerful platform to reconstruct life’s functions and shed light on the origin-of-life. More recently, they have been employed as convenient model systems to reconstitute membrane proteins, allowing their biochemical characterization and use in applications such as nanopore sequencing. While several techniques rely on commercially available phospholipids to form functional membranes, a grand challenge is the de novo construction of synthetic membranes in a manner that more closely mimics native lipid membrane generation in cells. Chemoselective coupling reactions have been used to generate cell-like synthetic lipid membranes. These methodologies suffer from distinct limitations, including the use of considerable amounts of micelle forming amphiphilic precursors that can interrupt lipid bilayer formation. Here we describe a novel de novo phospholipid synthesis strategy to rapidly form biomimetic membranes at physiological conditions in the micromolar concentration range, without the use of amphiphilic starting materials. The methodology takes advantage of oxime dialkylation via the condensation of two simple water-soluble precursors, a short-chain aldehyde and a dihydroxylamine-containing phosphocholine to afford a dual oxime phospholipid. Our strategy enables the chemoselective synthesis of phospholipids, which spontaneously self-assemble into membrane-bound vesicles. Membrane formation takes place in biologically relevant aqueous solution, and is stable in the presence of a variety of biomolecules and small polar molecules. Advantageously, we were able to reconstitute a membrane protein via in situ oxime phospholipid formation without the use of initial detergent solubilization. The oxime-based reconstitution technology can be used as a powerful tool for the straightforward fabrication of proteoliposomes, enabling thus to investigate membrane protein structure and function. We believe our novel chemoselective phospholipid synthesis approach will aid in creating functional artificial cells. Additionally, we envision our work will aid the study of membrane proteins within synthetic membranes or organelles