Deuterated natural phospholipids for advanced cellular membrane models

The cell membrane is a complex system, consisting of a semipermeable phospholipid bilayer embedded with various types of proteins, that controls the movement of substances in and out of a cell. Understanding the interactions between the cellular membrane and biomolecules, such as proteins, peptides and drugs, is thus highly important for both biomedical researchers and the pharmaceutical industry.

The sensitivity of neutrons to the biologically crucial hydrogen atoms, together with the powerful selective deuteration contrast method, makes neutron scattering techniques a particularly valuable probe for these experiments, providing information about the structure, dynamics and function.

Due to the thousands of different biomolecules studied in these experiments, it is more strategic to focus on deuterating the model membrane system, creating significant demand for the deuterated phospholipids needed to build them. The low availability of a number of biologically relevant phospholipid species is an important limitation. Furthermore, the partially deuterated phospholipids that are available – costing thousands of euros for a few milligrams – are all synthetic, compared to natural lipid molecules that enable the creation of a far superior model that is much more representative of a real cellular membrane.

With both world-class expertise and infrastructure, the Institut Laue-Langevin (ILL) has been a hub for protein deuteration for decades. Though initially the entire cellular system was deuterated, the protein extracted and anything remaining discarded, Giovanna Fragneto and Hanna Wacklin realised that every molecule present is highly valuable if it can be extracted and purified. An initiative thus began in 2013 to profile and separate the various molecules within the deuterated lipid mixture that includes phospholipids but also sterols and neutral lipids.

The achievement of this goal is far from straightforward and requires the consideration of a number of different aspects. First, the purity must be industry-grade if the phospholipids are to be used to build models for use in scientific experiments. The yield must also be high enough to justify the significant investment required for lipid deuteration. Finally, the lipids obtained must be of a high enough quality, without significant degradation, for them to be used to build model membrane systems.

Extraction and purification of various glycerophospholipid (GPL) mixtures from Pichia pastoris - Click to enlarge

“As a single purification step is insufficient, our idea was to combine two different methods,” explains Krishna Batchu, ILL scientist responsible for L-Lab, the lipid deuteration platform established to produce a wide range of pure natural phospholipid class mixtures in their deuterated forms for use in experiments carried out at the ILL.

“We employ a solid-phase extraction (SPE) column as the first step to remove the sterols and neutral lipids. The second additional step involves a high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) that enables the remaining polar mixture to be separated into different phospholipid families as a function of the polar headgroup.”

Significant research into existing protocols enabled the development of a robust and well-optimised system for the extraction and purification of various glycerophospholipid (GPL) mixtures from the yeast Picha Pastoris with a reproducibly high level of both purity and yield. The natural lipid mixtures, produced in both their hydrogenous and deuterated form, were then used to make advanced, more biologically relevant cell membrane models. The characterisation of these models using a number of different techniques demonstrated that although deuteration modified the lipid composition, the hydrogenous and deuterated models produced were both robust and structurally very comparable.

The produced membrane models were employed in experiments carried out at the ILL to study the interaction with COVID-19 particles, leading to publications in both the Journal of the American Chemical Society and Scientific Reports. “The results were robust and we got very good feedback,” explains Batchu. “We’re now getting enquiries from people around the world who are interested in procuring these natural lipids as there is no other neutron facility that has the ability to synthesise them.”

The success achieved at the L-Lab required the collaborative involvement of many different experts – including technicians, researchers, instrument scientists and PhD students, in addition to infrastructure across a number of different institutes. Expert guidance for the neutron experiments and data analysis was provided by Giovanna Fragneto (European Spallation Source) and Alessandra Luchini (University of Perugia). Experiments were carried out using instruments at both the ILL – small-angle neutron scattering (SANS) on D22 with the help of Lionel Porcar and Anne Martel, and neutron reflectometry (NR) on FIGARO with the help of Armando Maestro – and the European Synchrotron (ESRF) – small-angle X-ray scattering (SAXS) on BM29 with the help of Mark Tully.

Deuterated P. pastoris yeast cells were grown at the ILL’s deuteration facility, D-lab by Valerie Laux. The ILL/ESRF Partnership for Soft Condensed Matter (PSCM), under the guidance of Leonardo Chiappisi, provided the instrumentation necessary to perform quartz crystal microbalance with dissipation monitoring (QCM-D) measurements, while world-class expertise in gas chromatography-mass spectrometry (GC-MS) was provided by Cyrille Botté and Yoshiki Yamaryo-Botté at the Institute for Advanced Biosciences(IAB).

Another key element is CoruxFit, an open-source data analysis program built from scratch by Giacomo Corucci with valuable advice from Alessandra Luchini, Ernesto Scoppola (Max Planck Institute of Colloids and Interfaces), Francesco Spinozzi and Enrico Baldassarri (Università Politecnica delle Marche) and Moritz Frewein (Aix-Marseille University).

Corucci, now working as a postdoctoral researcher at Imperial College London, developed CoruxFit during his PhD at the ILL, jointly funded by the Université Grenoble Alpes (UGA). “Giacomo’s PhD was on a completely different project concerning phospholipases but he was so dedicated and motivated that he managed to advance both projects in parallel,” explains Batchu.

The scientists are now working to publish articles detailing CoruxFit – which has already been shared with other researchers to support their data analysis, in addition to results demonstrating the extension of the developed purification method to lipid extracts obtained from bacteria, in particular, E. coli. “There has been a huge rise in bacterial infections and there is thus significant interest in bacterial membrane systems in order to study their interaction with various antimicrobial peptides, drugs and molecules,” explains Batchu.

Initial work has also begun to continue advancing towards the ultimate goal: “Within each mixture there are several hundred different molecular species and what we want to be able to do is to separate out each of the individual molecular species from the complex mixture,” explains Batchu. The achievement of this goal would enable the creation of even more advanced cellular membrane models with properties that are both adjustable and controllable.

ILL support lab: L-Lab : Deuterated lipids at the ILL

Reference: Corucci, G., Batchu, K. C., et al. “Developing advanced models of biological membranes with hydrogenous and deuterated natural glycerophospholipid mixtures.” Journal of Colloid and Interface Science 645 (2023): 870-881.
https://doi.org/10.1016/j.jcis.2023.04.135

ILL Contact: Krishna Batchu