Crystallization of ApoA1 and ApoE4 Nanolipoprotein Particles and Initial XFEL-Based Structural Studies
Category
Published on
Type
journal-article
Author
Megan L. Shelby and Deepshika Gilbile and Thomas D. Grant and William J. Bauer and Brent Segelke and Wei He and Angela C. Evans and Natalia Crespo and Pontus Fischer and Tim Pakendorf and Vincent Hennicke and Mark S. Hunter and Alex Batyuk and Miriam Barthelmess and Alke Meents and Tonya L. Kuhl and Matthias Frank and Matthew A. Coleman
Citation
Shelby, M.L. et al., 2020. Crystallization of ApoA1 and ApoE4 Nanolipoprotein Particles and Initial XFEL-Based Structural Studies. Crystals, 10(10), p.886. Available at: http://dx.doi.org/10.3390/cryst10100886.
Abstract
Nanolipoprotein particles (NLPs), also called “nanodiscs”, are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via high-density lipoprotein (HDL) and low-density lipoprotein (LDL) maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics, and to develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment.
DOI
Funding
NSF-STC Biology with X-ray Lasers (NSF-1231306)
