Detergent-free extraction of a functional low-expressing GPCR from a human cell line

Dopamine receptors (DRs) are class A G-Protein Coupled Receptors (GPCRs) prevalent in the central nervous system (CNS). These receptors mediate physiological functions ranging from voluntary movement and reward recognition to hormonal regulation and hypertension. Drugs targeting dopaminergic neurotransmission have been employed to treat several neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, Huntington's disease, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. In vivo, incorporation of GPCRs into lipid membranes is known to be key to their biological function and, by inference, maintenance of their tertiary structure. A further significant challenge in the structural and biochemical characterization of human DRs is their low levels of expression in mammalian cells. Thus, the purification and enrichment of DRs whilst retaining their structural integrity and function is highly desirable for biophysical studies. A promising new approach is the use of styrene–maleic acid (SMA) copolymer to solubilize GPCRs directly in their native environment, to produce polymer-assembled Lipodisqs (LQs). We have developed a novel methodology to yield detergent-free D1-containing Lipodisqs directly from HEK293f cells expressing wild-type human dopamine receptor 1 (D1). We demonstrate that D1 in the Lipodisq retains activity comparable to that in the native environment and report, for the first time, the affinity constant for the interaction of the peptide neurotransmitter neurotensin (NT) with D1, in the native state

A new detergent-free approach to solubilize membrane proteins to maintain their native environment

Biophysical characterisation of membrane receptors in their native environment is essential for drug development, as lipid removal may affect conformational freedom and ligand binding. Membrane proteins are commonly solubilised with detergent for structural and functional studies, although the resulting micelles may be polydisperse, prone to aggregation and occlude of the binding site. Recently a hydrolysed copolymer of styrene and maleic acid (SMA) has been shown to be highly effective in extracting membrane proteins from native membranes without the use of detergent and the resulting coin-shaped nanoparticles, called Lipodisq (or SMALPs) are suitable for use in a wide range of biophysical methodologies. Here we have been able to purify and characterize a range of membrane proteins from their native environment including bacteriorhodopsin (bR), archaerhodopsin 3 (AR3), Dopamine receptor 1 (D1, a human GPCR) and EcMATE (a bacterial transporter) using size-exclusion and affinity chromatography, circular dichroism and dynamic light scattering. Microscale thermophoresis and radio-ligand binding have been used to assess the activity of the different proteins. These nanoparticles have further allowed us to characterize the oligomeric state and to identify native ligands by native mass spectrometry. The crystal structure of AR3 was obtained in the absence of detergent directly from its native membrane and its structure was solved to 1.3 Å resolution, where post-translational modifications, such as the pyroglutamate residue (PCA) at the N-terminus of AR3, and several side-chain rotamers are resolved for some key residues in the proton translocation channel as well as multiple water molecules. Some of the water molecules have partial occupancy within the extracellular-facing half channel, consistent with the existing FTIR data. Native mass spectrometry with Lipodisq has characterised the post-translational modifications carried by the protein. Moreover, a first structural study of the AR3 photointermediates using time-resolved XFEL crystallography has been performed. Lipodisq nanoparticles were used to study the lipid and protein composition of C. elegans by comparing the wild-type N2 worm to a mutant strain (agmo-1 ), which lacks for an important enzyme in the lipid biosynthesis. Finally, Lipodisqs have been evaluated as a drug delivery agent for Doxorubicin, a potent anti-tumour drug, which is commercially available as the PEGylated liposomal formulation Doxil. Previous work on the delivery of the drug to intracellular compartments of HeLa cells followed by confocal microscopy and IC50 measurements show that, unlike in the case of encapsulated systems, there is no decrease in in vitro efficacy over the water-solubilised form of the drug. Here the fluorescent derivative of the SMA polymer has been synthesised and its biodistribution followed in vivo.</p

A new detergent-free approach to solubilize membrane proteins to maintain their native environment

Biophysical characterisation of membrane receptors in their native environment is essential for drug development, as lipid removal may affect conformational freedom and ligand binding. Membrane proteins are commonly solubilised with detergent for structural and functional studies, although the resulting micelles may be polydisperse, prone to aggregation and occlude of the binding site. Recently a hydrolysed copolymer of styrene and maleic acid (SMA) has been shown to be highly effective in extracting membrane proteins from native membranes without the use of detergent and the resulting coin-shaped nanoparticles, called Lipodisq (or SMALPs) are suitable for use in a wide range of biophysical methodologies. Here we have been able to purify and characterize a range of membrane proteins from their native environment including bacteriorhodopsin (bR), archaerhodopsin 3 (AR3), Dopamine receptor 1 (D1, a human GPCR) and EcMATE (a bacterial transporter) using size-exclusion and affinity chromatography, circular dichroism and dynamic light scattering. Microscale thermophoresis and radio-ligand binding have been used to assess the activity of the different proteins. These nanoparticles have further allowed us to characterize the oligomeric state and to identify native ligands by native mass spectrometry. The crystal structure of AR3 was obtained in the absence of detergent directly from its native membrane and its structure was solved to 1.3 Ã resolution, where post-translational modifications, such as the pyroglutamate residue (PCA) at the N-terminus of AR3, and several side-chain rotamers are resolved for some key residues in the proton translocation channel as well as multiple water molecules. Some of the water molecules have partial occupancy within the extracellular-facing half channel, consistent with the existing FTIR data. Native mass spectrometry with Lipodisq has characterised the post-translational modifications carried by the protein. Moreover, a first structural study of the AR3 photointermediates using time-resolved XFEL crystallography has been performed. Lipodisq nanoparticles were used to study the lipid and protein composition of C. elegans by comparing the wild-type N2 worm to a mutant strain (agmo-1 ), which lacks for an important enzyme in the lipid biosynthesis. Finally, Lipodisqs have been evaluated as a drug delivery agent for Doxorubicin, a potent anti-tumour drug, which is commercially available as the PEGylated liposomal formulation Doxil. Previous work on the delivery of the drug to intracellular compartments of HeLa cells followed by confocal microscopy and IC50 measurements show that, unlike in the case of encapsulated systems, there is no decrease in in vitro efficacy over the water-solubilised form of the drug. Here the fluorescent derivative of the SMA polymer has been synthesised and its biodistribution followed in vivo.</p

From polymer chemistry to structural biology: The development of SMA and related amphipathic polymers for membrane protein extraction and solubilisation

Nanoparticles assembled with poly(styrene-maleic acid) copolymers, identified in the literature as Lipodisq, SMALPs or Native Nanodisc, are routinely used as membrane mimetics to stabilise protein structures in their native conformation. To date, transmembrane proteins of varying complexity (up to 8 beta strands or 48 alpha helices) and of a range of molecular weights (from 27&#x2009;kDa up to 500&#x2009;kDa) have been incorporated into this particle system for structural and functional studies. SMA and related amphipathic polymers have become versatile components of the biochemist's tool kit for the stabilisation, extraction and structural characterization of membrane proteins by techniques including cryo-EM and X-ray crystallography. Lipodisq formation does not require the use of conventional detergents and thus avoids their associated detrimental consequences. Here the development of this technology, from its fundamental concept and design to the diverse range of experimental methodologies to which it can now be applied, will be reviewed

Lipodisqs for eukaryote lipidomics with retention of viability: Sensitivity and resistance to Leucobacter infection linked to C.elegans cuticle composition.

Lipodisq™ nanoparticles have been used to extract surface lipids from the cuticle of two strains (wild type, N2 and the bacteria-resistant strain, agmo-1) of the C. elegans nematode without loss of viability. The extracted lipids were characterized by thin layer chromatography and MALDI-TOF-MS. The lipid profiles differed between the two strains. The extracted lipids from the bacteria-resistant strain, agmo-1, contained ether-linked (O-alkyl chain) lipids, in contrast to the wild-type strain which contained exclusively ester- linked (O-acyl) lipids. This observation is consistent with the loss of a functional alkylglycerol monooxygenase (AGMO) in the bacterial resistant strain agmo-1. The presence and abundance of other lipid species also differs between the wild-type N2 and agmo-1 nematodes, suggesting that the agmo-1 mutant strain attempts to compensate for the increase in ether-linked lipids by modulating other lipid-synthesis pathways. Together these differences not only affect the fragility of the cuticle and the buoyancy of the worm in aqueous buffer, but also interactions with surface-adhering bacteria. The much greater chemical stability of O-alkyl, non-hydrolysable linked lipids compared with hydrolysable O-acyl linked lipids, may be the origin of the resistance of the agmo-1 strain to bacterial infection, providing a more resilient cuticle for the nematode. Additionally, we show that lipid extraction with a polymer of styrene and maleic acid (SMA) provides a viable route to lipidomics studies with minimal perturbation of the organism

Lipodisqs for eukaryote lipidomics with retention of viability: Sensitivity and resistance to Leucobacter infection linked to C.elegans cuticle composition.

Lipodisq™ nanoparticles have been used to extract surface lipids from the cuticle of two strains (wild type, N2 and the bacteria-resistant strain, agmo-1) of the C. elegans nematode without loss of viability. The extracted lipids were characterized by thin layer chromatography and MALDI-TOF-MS. The lipid profiles differed between the two strains. The extracted lipids from the bacteria-resistant strain, agmo-1, contained ether-linked (O-alkyl chain) lipids, in contrast to the wild-type strain which contained exclusively ester- linked (O-acyl) lipids. This observation is consistent with the loss of a functional alkylglycerol monooxygenase (AGMO) in the bacterial resistant strain agmo-1. The presence and abundance of other lipid species also differs between the wild-type N2 and agmo-1 nematodes, suggesting that the agmo-1 mutant strain attempts to compensate for the increase in ether-linked lipids by modulating other lipid-synthesis pathways. Together these differences not only affect the fragility of the cuticle and the buoyancy of the worm in aqueous buffer, but also interactions with surface-adhering bacteria. The much greater chemical stability of O-alkyl, non-hydrolysable linked lipids compared with hydrolysable O-acyl linked lipids, may be the origin of the resistance of the agmo-1 strain to bacterial infection, providing a more resilient cuticle for the nematode. Additionally, we show that lipid extraction with a polymer of styrene and maleic acid (SMA) provides a viable route to lipidomics studies with minimal perturbation of the organism

Detergent-free Lipodisq nanoparticles facilitate high-resolution mass spectrometry of folded integral membrane proteins

Integral membrane proteins pose considerable challenges to mass spectrometry (MS) owing to the complexity and diversity of the components in their native environment. Here, we use native MS to study the post-translational maturation of bacteriorhodopsin (bR) and archaerhodopsin-3 (AR3), using both octyl-glucoside detergent micelles and lipid-based nanoparticles. A lower collision energy was required to obtain well-resolved spectra for proteins in styrene-maleic acid copolymer (SMA) Lipodisqs than in membrane scaffold protein (MSP) Nanodiscs. By comparing spectra of membrane proteins prepared using the different membrane mimetics, we found that SMA may favor selective solubilization of correctly folded proteins and better preserve native lipid interactions than other membrane mimetics. Our spectra reveal the correlation between the post-translation modifications (PTMs), lipid-interactions, and protein-folding states of bR, providing insights into the process of maturation of the photoreceptor proteins

Physicochemical characterization, toxicity and in vivo biodistribution studies of a discoidal, lipid-based drug delivery vehicle: Lipodisq nanoparticles containing doxorubicin

Many promising pharmaceutically active compounds have low solubility in aqueous environments and their encapsulation into efficient drug delivery vehicles is crucial to increase their bioavailability. Lipodisq nanoparticles are approximately 10 nm in diameter and consist of a circular phospholipid bilayer, stabilized by an annulus of SMA (a hydrolysed copolymer of styrene and maleic anhydride). SMA is used extensively in structural biology to extract and stabilize integral membrane proteins for biophysical studies. Here, we assess the potential of these nanoparticles as drug delivery vehicles, determining their cytotoxicity and the in vivo excretion pathways of their polymer and lipid components. Doxorubicin-loaded Lipodisqs were cytotoxic across a panel of cancer cell lines, whereas nanoparticles without the drug had no effect on cell proliferation. Intracellular doxorubicin release from Lipodisqs in HeLa cells occurred in the low-pH environment of the endolysosomal system, consistent with the breakdown of the discoidal structure as the carboxylate groups of the SMA polymer become protonated. Biodistribution studies in mice showed that, unlike other nanoparticles injected intravenously, most of the Lipodisq components were recovered in the colon, consistent with rapid uptake by hepatocytes and excretion into bile. These data suggest that Lipodisqs have the potential to act as delivery vehicles for drugs and contrast agents

Detergent-free extraction of a functional low-expressing GPCR from a human cell line

Dopamine receptors (DRs) are class A G-Protein Coupled Receptors (GPCRs) prevalent in the central nervous system (CNS). These receptors mediate physiological functions ranging from voluntary movement and reward recognition to hormonal regulation and hypertension. Drugs targeting dopaminergic neurotransmission have been employed to treat several neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, Huntington's disease, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. In vivo, incorporation of GPCRs into lipid membranes is known to be key to their biological function and, by inference, maintenance of their tertiary structure. A further significant challenge in the structural and biochemical characterization of human DRs is their low levels of expression in mammalian cells. Thus, the purification and enrichment of DRs whilst retaining their structural integrity and function is highly desirable for biophysical studies. A promising new approach is the use of styrene–maleic acid (SMA) copolymer to solubilize GPCRs directly in their native environment, to produce polymer-assembled Lipodisqs (LQs). We have developed a novel methodology to yield detergent-free D1-containing Lipodisqs directly from HEK293f cells expressing wild-type human dopamine receptor 1 (D1). We demonstrate that D1 in the Lipodisq retains activity comparable to that in the native environment and report, for the first time, the affinity constant for the interaction of the peptide neurotransmitter neurotensin (NT) with D1, in the native state