Aster-dependent nonvesicular transport facilitates dietary cholesterol uptake.

Intestinal absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1) assists in the initial step of dietary cholesterol uptake, but how cholesterol moves downstream of NPC1L1 is unknown. We show that Aster-B and Aster-C are critical for nonvesicular cholesterol movement in enterocytes. Loss of NPC1L1 diminishes accessible plasma membrane (PM) cholesterol and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate PM cholesterol and show endoplasmic reticulum cholesterol depletion. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, the Aster pathway can be targeted with a small-molecule inhibitor to manipulate cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption.

Aster-dependent non-vesicular transport facilitates dietary cholesterol uptake.

Intestinal cholesterol absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1), the target of the drug ezetimibe (EZ), assists in the initial step of dietary cholesterol uptake. However, how cholesterol moves downstream of NPC1L1 is unknown. Here we show that Aster-B and Aster-C are critical for non-vesicular cholesterol movement in enterocytes, bridging NPC1L1 at the plasma membrane (PM) and ACAT2 in the endoplasmic reticulum (ER). Loss of NPC1L1 diminishes accessible PM cholesterol in enterocytes and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate cholesterol at the PM and display evidence of ER cholesterol depletion, including decreased cholesterol ester stores and activation of the SREBP-2 transcriptional pathway. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, we show that the Aster pathway can be targeted with a small molecule inhibitor to manipulate dietary cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption. One-Sentence Summary: Identification of a targetable pathway for regulation of dietary cholesterol absorption.

Deuterated squalene and sterols from modified Saccharomyces cerevisiae.

Uniformly deuterated sterols and biosynthetically related materials are important for neutron, NMR, tracing and bioanalysis studies as well as critical tools for the creation of improved lipid nanoparticle formulations. The production of sufficient quantities of materials relies not only on the engineering of microorganisms to selectively accumulate desired materials but also methods for the isolation, purification and characterisation of these materials to ensure their usefulness. Uniformly deuterated squalene, the universal precursor to sterols in biological systems, has been produced and characterised. Cholesterol has been produced with controlled levels of uniform deuteration, increased biosynthetic yield and a methodology developed for the extraction and purification of this material without HPLC. Two sterols, not previously produced in deuterated forms, have been prepared with uniform deuteration: 22,23-dihydrobrassicasterol and 24-methylenecholesterol. This report triples the number of sterols that have been produced with uniform deuteration, purified and characterised and provides a silylation/silver ion chromatography protocol for the separation of sterols which differ by the degree of unsaturation. The techniques for the 13C NMR analysis of deuterated sterols, site-specific deuteration levels and an analysis of key biosynthetic steps based on these data are reported.

High-Density Lipoprotein function is modulated by the SARS-CoV-2 spike protein in a lipid-type dependent manner.

There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.

Investigation on the relationship between lipid composition and structure in model membranes composed of extracted natural phospholipids.

HYPOTHESIS: Unravelling the structural diversity of cellular membranes is a paramount challenge in life sciences. In particular, lipid composition affects the membrane collective behaviour, and its interactions with other biological molecules. EXPERIMENTS: Here, the relationship between membrane composition and resultant structural features was investigated by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry on in vitro membrane models of the mammalian plasma and endoplasmic-reticulum-Golgi intermediate compartment membranes in the form of Langmuir monolayers. Natural extracted yeast lipids were used because, unlike synthetic lipids, the acyl chain saturation pattern of yeast and mammalian lipids are similar. FINDINGS: The structure of the model membranes, orthogonal to the plane of the membrane, as well as their lateral packing, were found to depend strongly on their specific composition, with cholesterol having a major influence on the in-plane morphology, yielding a coexistence of liquid-order and liquid-disorder phases.

Deuteration for biological SANS: Case studies, success and challenges in chemistry and biology.

Small angle neutron scattering is a powerful complementary technique in structural biology. It generally requires, or benefits from, deuteration to achieve its unique potentials. Molecular deuteration has become a mature expertise, with deuteration facilities located worldwide to support access to the technique for a wide breadth of structural biology and life sciences. The sorts of problems well answered by small angle scattering and deuteration involve large (>10Å) scale flexible movements, and this approach is best used where high-resolution methods (crystallography, NMR, cryo-EM) leave questions unanswered. This chapter introduces deuteration, reviewing biological deuteration of proteins, lipids and sterols, and then steps through the ever-expanding range of deuterated molecules being produced by chemical synthesis and enabling sophisticated experiments using physiologically relevant lipids. Case studies of recent successful use of deuteration may provide illustrative examples for strategies for future experiments. We discuss issues of nomenclature for synthesised molecules of novel labeling and make recommendations for their naming. We reflect on our experiences, with cost associated with achieving an arbitrary deuteration level, and on the benefits of experimental co-design by user scientist, deuteration scientist, and neutron scattering scientist working together. Although methods for biological and chemical deuteration are published in the public domain, we recommend that the best method to deuterate is to engage with a deuteration facility.

The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from Mycobacterium ulcerans and Mycobacterium marinum.

The steroid binding CYP142 cytochrome P450 enzymes of Mycobacterium species are involved in the metabolism of cholesterol and its derivatives. The equivalent enzyme from Mycobacterium ulcerans was studied to compare the degree of functional conservation between members of this CYP family. We compared substrate binding of the CYP142A3 enzymes of M. ulcerans and M. marinum and CYP142A1 from M. tuberculosis using UV-vis spectroscopy. The catalytic oxidation of cholesterol derivatives by all three enzymes was undertaken. Both CYP142A3 enzymes were structurally characterized by X-ray crystallography. The amino acid sequences of the CYP142A3 enzymes are more similar to CYP142A1 from M. tuberculosis than CYP142A2 from Mycolicibacterium smegmatis. Both CYP142A3 enzymes have substrate binding properties, which are more resemblant to CYP142A1 than CYP142A2. The cholest-4-en-3-one-bound X-ray crystal structure of both CYP142A3 enzymes were determined at a resolution of <1.8 Å, revealing the substrate binding mode at a high level of detail. The structures of the cholest-4-en-3-one binding CYP142 enzymes from M. ulcerans and M. marinum demonstrate how the steroid binds in the active site of these enzymes. They provide an explanation for the high selectivity of the enzyme for terminal methyl C-H bond oxidation to form 26-hydroxy derivatives. These enzymes in pathogenic Mycobacterium species are candidates for inhibition. The work here demonstrates that similar drug molecules could target these CYP142 enzymes from different species in order to combat Buruli ulcer or tuberculosis.

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry.

SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.

Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Using Humidity to Control the Morphology and Properties of Electrospun BioPEGylated Polyhydroxybutyrate Scaffolds.

Electrospinning produces nanofibrous scaffolds with potential for tissue engineering and wound repair. Spinning parameters control scaffold morphology and properties. BioPEGylation of polyhydroxybutyrate (PHB) introduces terminal hydrophilic groups into the hydrophobic chain, making this natural-synthetic hybrid copolymer more susceptible to humidity. Varying the humidity from 10 to 50% RH during electrospinning had a relatively little effect on polyhydroxybutyrate (PHB) average fiber and pore diameters, which remained around 3.0 and 8.7 µm, respectively. In contrast, fiber and pore diameters for electrospun bioPEGylated PHB scaffolds varied significantly with humidity, peaking at 30% RH (5.5 and 14.1 µm, respectively). While scaffolds showed little change, hydrophobicity decreased linearly with humidity during electrospinning. Compared to solvent-cast films, electrospun scaffolds showed significantly greater average cell spread. A 108% increase for olfactory ensheathing cells (OECs) cultivated on bioPEGylated PHB scaffolds was proportionally greater than their counterparts on electrospun PHB scaffolds, (70%). OECS grown on BioPEGylated PHB scaffolds were over twice the size, 260 ± 20 µm diameter, than those on PHB electrospun scaffolds, 110 ± 18 µm diameter. Electrospun scaffolds also promoted cell health compared to their solvent-cast counterparts, with increases in the mitochondrial activity of 165 ± 13 and 196 ± 13% for PHB and bioPEGylated PHB, respectively. OECS cultivated on electrospun scaffolds of bioPEGylated PHB had significantly better membrane integrities compared to their counterparts on solvent-cast films, 47 ± 5% reducing to 17 ± 6%. The combination of bioPEGylation and humidity during electrospinning permitted significant controllable changes to scaffold morphology and properties. These changes resulted in the significantly greater promotion of cell growth on electrospun bioPEGylated PHB scaffolds compared to their solvent-cast counterparts and electrospun PHB.

Biopolymer deuteration for neutron scattering and other isotope-sensitive techniques.

The use of microbial biosynthesis to produced deuterated recombinant proteins is a well-established practice in investigations of the relationship between molecular structure and function using neutron scattering and nuclear magnetic resonance spectroscopy. However, there have been few reports of using microbial synthetic capacity to produce labeled native biopolymers. Here, we describe methods for the production of deuterated polyhydroxyalkanoate biopolyesters in bacteria, the polysaccharide chitosan in the yeast Pichia pastoris, and cellulose in the bacterium Gluconacetobacter xylinus. The resulting molecules offer not only multiple options in creating structural contrast in polymer blends and composites in structural studies but also insight into the biosynthetic pathways themselves.

Deuterated polymers for probing phase separation using infrared microspectroscopy.

Infrared (IR) microspectroscopy has the capacity to determine the extent of phase separation in polymer blends. However, a major limitation in the use of this technique has been its reliance on overlapping peaks in the IR spectra to differentiate between polymers of similar chemical compositions in blends. The objective of this study was to evaluate the suitability of deuteration of one mixture component to separate infrared (IR) absorption bands and provide image contrast in phase separated materials. Deuteration of poly(3-hydroxyoctanoate) (PHO) was achieved via microbial biosynthesis using deuterated substrates, and the characteristic C-D stretching vibrations provided distinct signals completely separated from the C-H signals of protonated poly(3-hydroxybutyrate) (PHB). Phase separation was observed in 50:50 (% w/w) blends as domains up to 100 µm through the film cross sections, consistent with earlier reports of phase separation observed by scanning electron microscopy (SEM) of freeze-fractured protonated polymer blends. The presence of deuterated phases throughout the film suggests there is some miscibility at smaller length scales, which increased with increasing PHB content. These investigations indicate that biodeuteration combined with IR microspectroscopy represents a useful tool for mapping the phase behavior of polymer blends.

BioPEGylation of polyhydroxybutyrate promotes nerve cell health and migration.

This study reports on the superior suitability of Polyhydroxybutyrate-polyethylene glycol hybrid polymers biosynthesised by Cupriavidus necator over PHB as biomaterials for tissue engineering. Incorporation of PEG106 (DEG) during PHB biosynthesis reduced crystallinity, molecular weight, and hydrophobicity while improving mechanical properties. In vitro olfactory ensheathing cell (OEC) proliferation was enhanced by cultivation on PHB-b-DEG films. Cultivation on PHB and PHB-b-DEG films showed no cytotoxic responses and cell viability and membrane integrity was sustained. PHB-b-DEG films promoted OECs entering into the DNA replication (S) phase and mitotic (G2-M) phase during the cell growth cycle and apoptosis was low. This study also confirmed an association between the level of neurite-outgrowth inhibitory protein (Nogo) and receptor pair Ig-like receptor B (PirB) expression and cell proliferation, both being down-regulated in cells grown on hybrid films when compared with PHB and asynchronous growth. Thus, DEG-terminated PHB-based biomaterials have great potential as biological scaffolds supporting nerve repair.

CAP37 activation of PKC promotes human corneal epithelial cell chemotaxis.

PURPOSE: The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. METHODS: Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 µM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr(505) phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively. RESULTS: Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr(505). Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. CONCLUSIONS: These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ.

Acute inhibition of fatty acid import inhibits GLUT4 transcription in adipose tissue, but not skeletal or cardiac muscle tissue, partly through liver X receptor (LXR) signaling.

OBJECTIVE: Insulin-mediated glucose uptake is highly sensitive to the levels of the facilitative GLUT protein GLUT4. Transcription of the GLUT4 gene is repressed in states of insulin deficiency and insulin resistance and can be induced by states of enhanced energy output, such as exercise. The cellular signals that regulate GLUT4 transcription are not well understood. We hypothesized that changes in energy substrate flux regulate GLUT4 transcription. RESEARCH DESIGN AND METHODS: To test this hypothesis, we used transgenic mice in which expression of the chloramphenicol acetyltransferase (CAT) gene is driven by a functional 895-bp fragment of the human GLUT4 promoter, thereby acting as a reporter for transcriptional activity. Mice were treated with a single dose of etomoxir, which inhibits the transport of long-chain fatty acids into mitochondria and increases basal, but not insulin-mediated, glucose flux. GLUT4 and transgenic CAT mRNA were measured. RESULTS: Etomoxir treatment significantly reduced CAT and GLUT4 mRNA transcription in adipose tissue, but did not change transcription in heart and skeletal muscle. Downregulation of GLUT4 transcription was cell autonomous, since etomoxir treatment of 3T3-L1 adipocytes resulted in a similar downregulation of GLUT4 mRNA. GLUT4 transcriptional downregulation required the putative liver X receptor (LXR) binding site in the human GLUT4 gene promoter in adipose tissue and 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the LXR agonist, TO901317, partially restored GLUT4 expression in etomoxir-treated cells. CONCLUSIONS: Our data suggest that long-chain fatty acid import into mitochondria in adipose tissue may produce ligands that regulate expression of metabolic genes.

Regulation of GLUT4 expression in denervated skeletal muscle.

Denervation by sciatic nerve resection causes decreased muscle glucose transporter 4 (GLUT4) expression, but little is known about the signaling events that cause this decrease. Experiments were designed to test the hypothesis that decreased GLUT4 expression in denervated muscle occurs because of decreased calcium/CaMK activity, which would then lead to decreased activation of the transcription factors myocyte enhancer factor 2 (MEF2) and GLUT4 enhancer factor (GEF), which are required for normal GLUT4 expression. GLUT4 mRNA was elevated in mice expressing constitutively active CaMK isoform IV (CaMKIV) and decreased by denervation. Denervation decreased GEF binding to the promoter and the content of GEF in the nucleus, but there was no change in either MEF2 binding or MEF2 protein content. Expression of a MEF2-dependent reporter gene did not change in denervated skeletal muscle. To determine the domains of the GLUT4 promoter that respond to denervation, transgenic mice expressing the chloramphenicol acetyl transferase (CAT) reporter gene driven by different lengths of the human GLUT4 promoter were denervated. Using several different promoter/reporter gene constructs, we found that all areas of the GLUT4 promoter were truncated or missing, except for the MEF2 binding domain and the basal promoter. All of the GLUT4 promoter/CAT reporter constructs evaluated responded normally to denervation. Our data lead us to conclude that decreased CaMK activity is not the reason for decreased GLUT4 content in denervated muscle and that negative control of GLUT4 expression is not mediated through the MEF2 or GEF-binding domains. These findings indicate that withdrawal of a GEF- or MEF2-dependent signal is not likely a major determinant of the denervation effect on GLUT4 expression. Thus, the response to denervation may be mediated by other elements present in the basal promoter of the GLUT4 gene.

In vivo deuteration strategies for neutron scattering analysis of bacterial polyhydroxyoctanoate.

The cultivation of microorganisms on deuterated substrates has allowed us to control deuterium incorporation into biopolymer systems which is important for characterisation using neutron scattering techniques. Bacterial polyhydroxyoctanoate (PHO) is a polyester formed within inclusions inside bacterial cells and was deuterated in vivo under various conditions to characterise the formation of these inclusions by neutron scattering. Manipulation of deuterated media during microbial growth and PHO production phases resulted in polymer with partial or complete substitution of hydrogen by deuterium, as shown by gas chromatography. Sequential feeding of hydrogenated and deuterated forms of the same precursor was used to demonstrate that neutron scattering analysis could be used to differentiate between chemically similar phases in these polymer inclusions.

Production and use of deuterated polyhydroxyoctanoate in structural studies of PHO inclusions.

This work reports on the biosynthesis of polyhydroxyalkanoates with medium chain length alkyl substituents in the side chain by Pseudomonas oleovorans using hydrogenated and deuterated substrates. These investigations aimed to obtain polyhydroxyalkanoates with varying degrees of deuterium substitution, and establish whether they are suitable analogues for structural investigation. In order to understand the formation and structure of inclusions in their native state, whole inclusions were isolated from microbial cells and were analysed using Small Angle Neutron Scattering. A contrast variation study was conducted on hydrogenated and deuterated inclusions of polyhydroxyoctanoate, as well as inclusions resulting from co-feeding or sequentially feeding different precursors. The data indicated a core/shell structure resulting from feeding hydrogenated followed by perdeuterated PHO precursor, and demonstrated the utility of this analysis for characterising chemically similar systems.

Biosynthesis and characterization of deuterated polyhydroxyoctanoate.

The synthesis of a polyhydroxyalkanoate with medium chain length alkyl substituents by Pseudomonas oleovoranswas investigated using protonated and deuterated forms of octanoic acid in a minimal salts medium. Cultivation with deuterated octanoic acid resulted in a reduced rate of polymer accumulation compared to that with its protonated counterpart (107 and 207 mg of polymer L(-1) of medium h(-1) of cultivation, respectively). Nuclear magnetic resonance and gas chromatography coupled mass spectrometry of the derivatized polymer was used to establish the extent and distribution of deuterium in the biopolymer. A partially deuterated heteropolymer with 3-hydroxyoctanoic acid as the main constituent was produced. Deuteration is an important tool for contrast variation studies using neutron scattering, but predicates that the deuterated polymer is otherwise comparable in its physiochemical and material properties to its protonated counterpart. In studies reported here, the deuterated biopolymer exhibited an additional diffraction maximum at 7.55 A and slight differences in its melting point (60 and 55 degrees C) and glass transition temperature (-39 and -36 degrees C) when compared to its protonated equivalent. While significant differences between the protonated and deuterated biopolymers were determined, our results support the use of this deuterated polyhydroxyalkanoate in its application in investigations using analytical neutron scattering techniques.