January 1, 2019.
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).
Biochemistry | Biology | Biotechnology | Medicine and Health Sciences | Molecular Biology | Structural Biology
Dr. Megan Shelby
Lawrence Livermore National Laboratory (LLNL)
The 2019 STEM Teacher and Researcher Program and this project have been made possible through support from Chevron (www.chevron.com), the National Marine Sanctuary Foundation (www.marinesanctuary.org), the National Science Foundation through the Robert Noyce Program under Grant #1836335 and 1340110, the California State University Office of the Chancellor, and California Polytechnic State University in partnership with Lawrence Livermore National Laboratory and University of Oregon Noyce ESPRIT, funded by NSF DUE 1660724. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the funders. This work was also supported by funds from the following: National Institute of Health: NIGMS grant R01GM117342 and NIAID grant R21AI120925. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.