Crystallization of ApoA1 and ApoE4 nanolipoprotein particles and initial XFEL-based structural studies.

Megan Shelby,Angela Evans,Alke Meents,Miriam Barthelmess,Tonya Kuhl,Matthew Coleman,Wei He,Deepshika Gilbile,Mark Hunter,Matthias Frank,William Bauer,Brent Segelke,Thomas Grant,Natalia Crespo,Alex Batyuk,Pontus Fischer,Tim Pakendorf,Vincent Hennicke

doi: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 HDL and 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 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 in characteristics 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.

Highlights

Nanolipoprotein particles (NLPs), or “nanodiscs”, are nanoscale complexes with apolipoproteins forming a corralled, discoidal bilayer [1]
To enable fixed target SFX in-vacuum, we have recently demonstrated the use of large-area few-layer graphene (FLG)
Protein yields ranged from 3–4 mg of apolipoprotein, which are typical for a 1 mL cell-free reaction

Summary

Introduction

Nanolipoprotein particles (NLPs), or “nanodiscs”, are nanoscale complexes with apolipoproteins forming a corralled, discoidal bilayer [1]. Discoidal lipoproteins, which are closely related to NLPs, represent a critical physiological transition state from lipid-free apolipoprotein to spherical HDL and LDL particles during HDL/LDL maturation, and play a role in cardiovascular disease risk, protection against atherosclerosis, and amyloid related diseases due to their key role in reverse cholesterol transport (RCT) [16,17]. Interaction between nascent HDL/LDL discs and lecithin cholesterol acyltransferase (LCAT). Current structural models of the disc suggest an anti-parallel apolipoprotein chain orientation surrounding the lipid bilayer patch that can adjust to accommodate a number of discrete particle diameters based on the protein to lipid ratio. Computational modeling has been performed in the presence of phospholipids [24] and several NMR solution structures exist [24,25,26]

Methods

Results

Conclusion