Membrane proteins make up approximately 30% of the cellular proteome and account for over 60% of pharmaceutical targets.1 Determining the structures of this class of proteins is critical to our understanding of disease states and will advance rational drug design. But membrane proteins have limited solubility, rarely form large crystals that diffract well, and often misfold outside of a bilayer, hindering crystallographic studies.1 Nanolipoprotein particles (NLPs) have arisen as a platform to readily solubilize membrane proteins while mimicking a native lipid environment. NLPs consist of a discoidal phospholipid bilayer encircled by an apolipoprotein belt. In an effort to optimize and improve crystallization of empty NLPs, we altered the fluidity of the lipid bilayer by incorporating photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Here, we used a cell-free expression system with apolipoprotein A1 (ApoA1) plasmid and micellar lipids to assemble NLPs. Based on high throughput crystallization screening data, we reproduced and validated crystallization conditions for X-NLPs and optimized conditions for non-crosslinked NLPs (DMPC NLPs)
