Research Projects

Membrane proteins (MPs) are key to many fundamental processes in cells. Impairment of structure and functions of MPs is linked to many disease states. Therefore, it is immensely important to carry out structural and biophysical investigations on MPs. Progress in MP biology is intimately linked to advancement in protein expression methodologies and the development of molecular tools that are essential for stabilizing MP structures. In this proposal, we aim to combine the utilities of lipid sponge phase droplets and cell-free protein synthesis technologies to advance basic research on MPs. This highly interdisciplinary project will combine organic synthesis, biochemistry, molecular biology, microscopy, and soft matter physics. Lipid sponge droplets are a class of self-assembled material comprising of a three-dimensional network of lipid bilayers interspersed by nanometric aqueous channels. The droplets can spontaneously sequester high concentrations of MPs within the bilayer network. The droplets undergo liquid-liquid phase separation – a property that can be exploited for purification of sequestered MPs. Effectively, lipid sponge droplets combine the utilities of membrane-mimetic systems like liposomes, and bio-separation materials like affinity beads or chromatographic resins. Till date, there are only a handful of lipids/amphiphiles that are known to form sponge phase droplets. Also, systematic studies relating structures of lipids to their propensities to form sponge phase droplets are extremely rare. Therefore, we will first focus on building a comprehensive toolbox of novel sponge phase-forming lipids/amphiphiles via rational molecular design and organic synthesis. The physical properties of the novel sponge phases will be thoroughly characterized by small-angle X-ray scattering (aqueous pore size), rheology (viscosity), fluorescence spectroscopy (behavior of fluorophores), cryo-SEM (internal morphology), differential scanning calorimetry (thermal phase transition), and thermogravimetric analysis (water content). After gaining deep insight into the material properties of the novel sponge phases, they will be coupled with cell-free synthesis of MPs. We will express several prokaryotic and eukaryotic MPs in cell-free systems in the presence of droplets, measure the levels of protein expression, and assay for their function. Finally, we will lay the groundwork for carrying out micro- and nano-crystallization of MPs in the sponge phases which will be useful for cutting-edge structural techniques like microelectron diffraction and serial femtosecond crystallography. If successful, we will be able to establish lipid sponge droplets as a new platform technology to accelerate MP research. This platform will enable rapid, small-scale, economic (both material and cost-wise), and high-throughput production of reconstituted membrane proteins which will be directly used to prepare samples (for example, crystals) for advanced structural techniques.