Novel Stilbene-Nitroxyl Hybrid Compounds Display Discrete Modulation of Amyloid Beta Toxicity and Structure.

Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These "toxic oligomers" have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AßO) and the oxidative stress elicited by AßO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer's disease in a powerful, synergistic manner. Examination of AßO structure by various biophysical tools shows that each stilbene candidate uniquely alters AßO conformation and toxicity, providing insight towards the future development of structural correctors for AßO. Correlations of AßO structural modulation and bioactivity displayed by each provides insights for future testing in vivo. The multi-target activity of these hybrid molecules represents a highly advantageous feature for disease modification in Alzheimer's, which displays a complex, multifactorial etiology. Importantly, these novel small molecules intervene with intraneuronal AßO, a necessary feature to counter the cycle of dysregulation, oxidative stress and inflammation triggered during the earliest stages of disease progression.

Determining structure and action mechanism of LBF14 by molecular simulation.

The recently discovered, membrane-active peptide LBF14 contains several non-proteinogenic amino acids and is able to transform vesicles into tubule networks. The exact membrane interaction mechanism and detailed secondary structure are yet to be determined. We performed molecular dynamics simulations of LBF14 and let it fold de novo into its ensemble of native secondary structures. Histidine protonation state effects on secondary structure were investigated. An MD simulation of the peptide with a lipid bilayer was performed. Simulation results were compared to circular dichroism and electron paramagnetic resonance data of previous studies. LBF14 contains a conserved helical section in an otherwise random structure. Helical stability is influenced by histidine protonation. The peptide localized to the polar layer of the membrane, consistent with experimental results. While the overall secondary structure is unaffected by membrane interaction, Ramachandran plot analysis yielded two distinct peptide conformations during membrane interaction. This conformational change was accompanied by residue repositioning within the membrane. LBF14 only affected the local order in the membrane, and had no measurable effect on pressure. The simulation results are consistent with the previously proposed membrane interaction mechanism of LBF14 and can additionally explain the local interaction mechanism. Communicated by Ramaswamy H. Sarma.

Identification of amyloid beta in small extracellular vesicles via Raman spectroscopy.

One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and deposition of amyloid-beta (Aß) peptide into extracellular plaques. Existing research indicates that extracellular vesicles (EVs) can carry Aß associated with AD. However, characterization of the EVs-associated Aß and its conformational variants has yet to be realized. Raman spectroscopy is a label-free and non-destructive method that is able to assess the biochemical composition of EVs. This study reports for the first time the Raman spectroscopic fingerprint of the Aß present in the molecular cargo of small extracellular vesicles (sEVs). Raman spectra were measured from sEVs isolated from Alzheimer's disease cell culture model, where secretion of Aß is regulated by tetracycline promoter, and from midbrain organoids. The averaged spectra of each sEV group showed considerable variation as a reflection of the biochemical content of sEVs. Spectral analysis identified more intense Raman peaks at 1650 cm-1 and 2930 cm-1 attributable to the Aß peptide incorporated in sEVs produced by the Alzheimer's cell culture model. Subsequent analysis of the spectra by principal component analysis differentiated the sEVs of the Alzheimer's disease cell culture model from the control groups of sEVs. Moreover, the results indicate that Aß associated with secreted sEVs has a α-helical secondary structure and the size of a monomer or small oligomer. Furthermore, by analyzing the lipid content of sEVs we identified altered fatty acid chain lengths in sEVs that carry Aß that may affect the fluidity of the EV membrane. Overall, our findings provide evidence supporting the use of Raman spectroscopy for the identification and characterization of sEVs associated with potential biomarkers of neurological disorders such as toxic proteins.

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

Application of a benchtop colorimetric method for quantification of blood propofol levels.

Quantification of plasma propofol (2,6-diisopropylphenol) in the context of clinical anaesthesia is challenging because of the need for offline blood sample processing using specialised laboratory equipment and techniques. In this study we sought to refine a simple procedure using solid phase extraction and colorimetric analysis into a benchtop protocol for accurate blood propofol measurement. The colorimetric method based on the reaction of phenols (e.g. propofol) with Gibbs reagent was first tested in 10% methanol samples (n = 50) containing 0.5-6.0 µg/mL propofol. Subsequently, whole blood samples (n = 15) were spiked to known propofol concentrations and processed using reverse phase solid phase extraction (SPE) and colorimetric analysis. The standard deviation of the difference between known and measured propofol concentrations in the methanol samples was 0.11 µg/mL, with limits of agreement of - 0.21 to 0.22 µg/mL. For the blood-processed samples, the standard deviation of the difference between known and measured propofol concentrations was 0.09 µg/mL, with limits of agreement - 0.18 to 0.17 µg/mL. Quantification of plasma propofol with an error of less than 0.2 µg/mL is achievable with a simple and inexpensive benchtop method.

Superhydrophobic bowl-like SERS substrates patterned from CMOS sensors for extracellular vesicle characterization.

Using a regular CMOS sensor as a template, we are able to fabricate a simple but highly effective superhydrophobic SERS substrate. Specifically, we decorated the microlens layer of the sensor with 7 µm polystyrene beads to obtain a PDMS patterned replica. The process resulted in a uniform pattern of voids in the PDMS (denoted nanobowls) that are intercalated with a few larger voids (denoted here microbowls). The voids act as superhydrophobic substrates with analyte concentration capabilities in bigger bowl-like structures. Silver nanoparticles were directly grown on the patterned PDMS substrate inside both the nano- and microbowls, and serve as strong electromagnetic field enhancers for the SERS substrate. After systematic characterization of the fabricated SERS substrate by atomic force microscopy and scanning electron microscopy, we demonstrated its SERS performance using 4-aminothiophenol as a reporter molecule. Finally, we employed this innovative substrate to concentrate and analyze extracellular vesicles (EVs) isolated from an MC65 neural cell line in an ultralow sample volume. This substrate can be further exploited for the investigation of various EV biomarkers for early diagnosis of different diseases using liquid biopsy.

Surface-enhanced Raman scattering sensing platform for detecting amyloid-ß peptide interaction with an aggregation inhibitor.

Soluble, small amyloid-ß oligomers (AßO) are recognized as significant contributors to the pathology of Alzheimer's disease (AD). Although drugs for treating AD symptoms have been approved, no therapy targeting amyloid-ß (Aß) capable of modifying the course of the disease is available. In an effort to develop a label-free method for screening new anti-AD therapeutic agents, we show the use of a surface-enhanced Raman scattering (SERS) active substrate for detecting the interactions between Aß peptides and spin-labeled fluorine (SLF), a peptide aggregation inhibitor. Changes in the peak positions and intensity ratios of two spectral peaks near 1600cm-1 and 2900cm-1 can be used to monitor the molecular interactions between SLF and Aß. This study demonstrates the potential of SERS spectroscopy for rapidly screening and identifying new anti-Aß therapeutic agents.

Discovery and mechanistic characterization of a structurally-unique membrane active peptide.

Membrane active peptides (MAPs) have gained wide interest due to their far reaching applications in drug discovery and drug delivery. The search for new MAPs, however, has been largely skewed with bias selecting for physicochemical parameters believed to be important for membrane activity, such as alpha helicity, cationicity and hydrophobicity. Here we carry out a search-and-find strategy to screen a 100,000-membered one-bead-one-compound (OBOC) combinatorial peptide library for lead compounds, agnostic of those physicochemical constraints. Such a synthetic strategy also permits expansion of our peptide repertoire to include unnatural amino acids. Using this approach, we discovered a structurally unique lead peptide LBF14, a linear 14-mer peptide, that induces gross morphological disruption of membranes, irrespective of membrane composition. Further, we demonstrate that the unique insertion mechanism of the peptide, visualized by spinning disc confocal microscopy and further analyzed by electron paramagnetic resonance measurements, may be the cause of this large scale membrane deformation. We also demonstrate the robustness, reproducibility, and potential application of this technique to discover and characterize new membrane active peptides that display activity by local insertion and subsequent allosteric effects leading to global membrane disruption.

Real-time measurements of milk fat globule membrane modulation during simulated intestinal digestion using electron paramagnetic resonance spectroscopy.

Milk Fat Globules with their unique interfacial structure and membrane composition are a key nutritional source for mammalian infants, however, there is a limited understanding of the dynamics of fat digestion in these structures. Lipid digestion is an interfacial process involving interactions of enzymes and bile salts with the interface of suspended lipid droplets in an aqueous environment. In this study, we have developed an electron paramagnetic resonance spectroscopy approach to evaluate real time dynamics of milk fat globules interfacial structure during simulated intestinal digestion. To measure these dynamics, natural milk fat globule membrane was labeled with EPR-active probe, partitioning of EPR probes into MFGs membrane was validated using saturation-recovery measurements and calculation of the depth parameter Φ. After validation, the selected spin probe was used to evaluate the membrane's fluidity as a measure of the interface's modulation in the presence of bile salts and pancreatic lipase. Independently, bile salts were found to have a rigidifying effect on the spin probed MFGM, while pancreatic lipase resulted in an increase in membrane fluidity. When combined, the effect of lipase appears to be diminished in the presence of bile salts. These results indicate the efficacy of EPR in providing an insight into small time scale molecular dynamics of phospholipid interfaces in milk fat globules. Understanding interfacial dynamics of naturally occurring complex structures can significantly aid in understanding the role of interfacial composition and structural complexity in delivery of nutrients during digestion.

Completion of the Vimentin Rod Domain Structure Using Experimental Restraints: A New Tool for Exploring Intermediate Filament Assembly and Mutations.

Electron paramagnetic resonance (EPR) spectroscopy of full-length vimentin and X-ray crystallography of vimentin peptides has provided concordant structural data for nearly the entire central rod domain of the protein. In this report, we use a combination of EPR spectroscopy and molecular modeling to determine the structure and dynamics of the missing region and unite the separate elements into a single structure. Validation of the linker 1-2 (L1-2) modeling approach is demonstrated by the close correlation between EPR and X-ray data in the previously solved regions. Importantly, molecular dynamic (MD) simulation of the constructed model agrees with spin label motion as determined by EPR. Furthermore, MD simulation shows L1-2 heterogeneity, with a concerted switching of states among the dimer chains. These data provide the first ever experimentally driven model of a complete intermediate filament rod domain, providing research tools for further modeling and assembly studies.

Mitochondrial oxidative stress-induced transcript variants of ATF3 mediate lipotoxic brain microvascular injury.

Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for cardiovascular disease and vascular dementia (VaD). Accumulating evidence indicates that both atherosclerosis and VaD are linked to vascular inflammation. However, the role of TGRL in vascular inflammation, which increases risk for VaD, remains largely unknown and its underlying mechanisms are still unclear. We strived to determine the effects of postprandial TGRL exposure on brain microvascular endothelial cells, the potential risk factor of vascular inflammation, resulting in VaD. We showed in Aung et al., J Lipid Res., 2016 that postprandial TGRL lipolysis products (TL) activate mitochondrial reactive oxygen species (ROS) and increase the expression of the stress-responsive protein, activating transcription factor 3 (ATF3), which injures human brain microvascular endothelial cells (HBMECs) in vitro. In this study, we deployed high-throughput sequencing (HTS)-based RNA sequencing methods and mito stress and glycolytic rate assays with an Agilent Seahorse XF analyzer and profiled the differential expression of transcripts, constructed signaling pathways, and measured mitochondrial respiration, ATP production, proton leak, and glycolysis of HBMECs treated with TL. Conclusions: TL potentiate ROS by mitochondria which activate mitochondrial oxidative stress, decrease ATP production, increase mitochondrial proton leak and glycolysis rate, and mitochondria DNA damage. Additionally, CPT1A1 siRNA knockdown suppresses oxidative stress and prevents mitochondrial dysfunction and vascular inflammation in TL treated HBMECs. TL activates ATF3-MAPKinase, TNF, and NRF2 signaling pathways. Furthermore, the NRF2 signaling pathway which is upstream of the ATF3-MAPKinase signaling pathway, is also regulated by the mitochondrial oxidative stress. We are the first to report differential inflammatory characteristics of transcript variants 4 (ATF3-T4) and 5 (ATF3-T5) of the stress responsive gene ATF3 in HBMECs induced by postprandial TL. Specifically, our data indicates that ATF3-T4 predominantly regulates the TL-induced brain microvascular inflammation and TNF signaling. Both siRNAs of ATF3-T4 and ATF3-T5 suppress cells apoptosis and lipotoxic brain microvascular endothelial cells. These novel signaling pathways triggered by oxidative stress-responsive transcript variants, ATF3-T4 and ATF3-T5, in the brain microvascular inflammation induced by TGRL lipolysis products may contribute to pathophysiological processes of vascular dementia.

A Bifunctional Anti-Amyloid Blocks Oxidative Stress and the Accumulation of Intraneuronal Amyloid-Beta.

There is growing recognition regarding the role of intracellular amyloid beta (Aß) in the Alzheimer's disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aß likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aß and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aß accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aß. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aß. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aß-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aß targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aß, its promotion of ROS, and Aß metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aß oligomer formation.

Oligomerization Alters Binding Affinity Between Amyloid Beta and a Modulator of Peptide Aggregation.

The soluble oligomeric form of the amyloid beta (Aß) peptide is the major causative agent in the molecular pathogenesis of Alzheimer's disease (AD). We have previously developed a pyrroline-nitroxyl fluorene compound (SLF) that blocks the toxicity of Aß. Here we introduce the multi-parametric surface plasmon resonance (MP-SPR) approach to quantify SLF binding and effect on the self-association of the peptide via a label-free, real-time approach. Kinetic analysis of SLF binding to Aß and measurements of layer thickness alterations inform on the mechanism underlying the ability of SLF to inhibit Aß toxicity and its progression towards larger oligomeric assemblies. Depending on the oligomeric state of Aß, distinct binding affinities for SLF are revealed. The Aß monomer and dimer uniquely possess sub-nanomolar affinity for SLF via a non-specific mode of binding. SLF binding is weaker in oligomeric Aß, which displays an affinity for SLF on the order of 100 µM. To complement these experiments we carried out molecular docking and molecular dynamics simulations to explore how SLF interacts with the Aß peptide. The MP-SPR results together with in silico modeling provide affinity data for the SLF-Aß interaction and allow us to develop a new general method for examining protein aggregation.

A Metal-Free Method for Producing MRI Contrast at Amyloid-ß.

Alzheimer's disease (AD) is characterized by depositions of the amyloid-ß (Aß) peptide in the brain. The disease process develops over decades, with substantial neurological loss occurring before a clinical diagnosis of dementia can be rendered. It is therefore imperative to develop methods that permit early detection and monitoring of disease progression. In addition, the multifactorial pathogenesis of AD has identified several potential avenues for AD intervention. Thus, evaluation of therapeutic candidates over lengthy trial periods also demands a practical, noninvasive method for measuring Aß in the brain. Magnetic resonance imaging (MRI) is the obvious choice for such measurements, but contrast enhancement for Aß has only been achieved using Gd(III)-based agents. There is great interest in gadolinium-free methods to image the brain. In this study, we provide the first demonstration that a nitroxide-based small-molecule produces MRI contrast in brain specimens with elevated levels of Aß. The molecule is comprised of a  fluorene (a molecule with high affinity for Aß) and a nitroxide spin label (a paramagnetic MRI contrast species). Labeling of brain specimens with the spin-labeled fluorene produces negative contrast in samples from AD model mice whereas no negative contrast is seen in specimens harvested from wild-type mice. Injection of spin-labeled fluorene into live mice resulted in good brain penetration, with the compound able to generate contrast 24-h post injection. These results provide a proof of concept method that can be used for early, noninvasive, gadolinium-free detection of amyloid plaques by MRI.

Molecular crowding impacts the structure of apolipoprotein A-I with potential implications on in vivo metabolism and function.

The effect molecular crowding, defined as the volume exclusion exerted by one soluble inert molecule upon another soluble molecule, has on the structure and self-interaction of lipid-free apoA-I were explored. The influence of molecular crowding on lipid-free apoA-I oligomerization and internal dynamics has been analyzed using electron paramagnetic resonance (EPR) spectroscopy measurements of nitroxide spin label at selected positions throughout the protein sequence and at varying concentrations of the crowding agent Ficoll-70. The targeted positions include sites previously shown to be sensitive for detecting intermolecular interaction via spin-spin coupling. Circular dichroism was used to study secondary structural changes in lipid-free apoA-I imposed by increasing concentrations of the crowding agent. Crosslinking and SDS-PAGE gel analysis was employed to further characterize the role molecular crowding plays in inducing apoA-I oligomerization. It was concluded that the dynamic apoA-I structure and oligomeric state was altered in the presence of the crowding agent. It was also found that the C-terminal was slightly more sensitive to molecular crowding. Finally, the data described the region around residue 217 in the C-terminal domain of apoA-I as the most sensitive reporter of the crowding-induced self-association of apoA-I. The implications of this behavior to in vivo functionality are discussed. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 683-692, 2016.

Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways.

Dysfunction of the cerebrovasculature plays an important role in vascular cognitive impairment (VCI). Lipotoxic injury of the systemic endothelium in response to hydrolyzed triglyceride-rich lipoproteins (TGRLs; TGRL lipolysis products) or a high-fat Western diet (WD) suggests similar mechanisms may be present in brain microvascular endothelium. We investigated the hypothesis that TGRL lipolysis products cause lipotoxic injury to brain microvascular endothelium by generating increased mitochondrial superoxide radical generation, upregulation of activating transcription factor 3 (ATF3)-dependent inflammatory pathways, and activation of cellular oxidative stress and apoptotic pathways. Human brain microvascular endothelial cells were treated with human TGRL lipolysis products that induced intracellular lipid droplet formation, mitochondrial superoxide generation, ATF3-dependent transcription of proinflammatory, stress response, and oxidative stress genes, as well as activation of proapoptotic cascades. Male apoE knockout mice were fed a high-fat/high-cholesterol WD for 2 months, and brain microvessels were isolated by laser capture microdissection. ATF3 gene transcription was elevated 8-fold in the hippocampus and cerebellar brain region of the WD-fed animals compared with chow-fed control animals. The microvascular injury phenotypes observed in vitro and in vivo were similar. ATF3 plays an important role in mediating brain microvascular responses to acute and chronic lipotoxic injury and may be an important preventative and therapeutic target for endothelial dysfunction in VCI.

How to Study Intermediate Filaments in Atomic Detail.

Studies of the intermediate filament (IF) structure are a prerequisite of understanding their function. In addition, the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs. Over the years, considerable progress has been made on the atomic structure of the elementary building block of all IFs, the coiled-coil dimer. Here, we discuss the approaches, methods and practices that have contributed to this advance. With abundant genetic information on hand, bioinformatics approaches give important insights into the dimer structure, including the head and tail regions poorly assessable experimentally. At the same time, the most important contribution has been provided by X-ray crystallography. Following the "divide-and-conquer" approach, many fragments from several IF proteins could be crystallized and resolved to atomic resolution. We will systematically cover the main procedures of these crystallographic studies, suggest ways to maximize their efficiency, and also discuss the possible pitfalls and limitations. In addition, electron paramagnetic resonance with site-directed spin labeling was another method providing a major impact toward the understanding of the IF structure. Upon placing the spin labels into specific positions within the full-length protein, one can evaluate the proximity of the labels and their mobility. This makes it possible to make conclusions about the dimer structure in the coiled-coil region and beyond, as well as to explore the dimer-dimer contacts.

Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure.

Alzheimer's disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aß) peptides. Soluble oligomers of the Aß peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aß oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aß) prevent and disrupt oligomeric assemblies of Aß in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aß shows a continuous, progressive change over a 24-hour period, while the spectrum of Aß treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aß within the oligomer provides a complementary determinant of Aß toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aß, it does induce a net reduction in beta secondary content compared to untreated samples of Aß. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aß oligomers and disrupt existing oligomers, while retaining Aß as a population of smaller, yet largely disordered oligomers.

Spectroscopic Characterization of Structural Changes in Membrane Scaffold Proteins Entrapped within Mesoporous Silica Gel Monoliths.

The changes in the orientation and conformation of three different membrane scaffold proteins (MSPs) upon entrapment in sol-gel-derived mesoporous silica monoliths were investigated. MSPs were examined in either a lipid-free or a lipid-bound conformation, where the proteins were associated with lipids to form nanolipoprotein particles (NLPs). NLPs are water-soluble, disk-shaped patches of a lipid bilayer that have amphiphilic MSPs shielding the hydrophobic lipid tails. The NLPs in this work had an average thickness of 5 nm and diameters of 9.2, 9.7, and 14.8 nm. We have previously demonstrated that NLPs are more suitable lipid-based structures for silica gel entrapment than liposomes because of their size compatibility with the mesoporous network (2-50 nm) and minimally altered structure after encapsulation. Here we further elaborate on that work by using a variety of spectroscopic techniques to elucidate whether or not different MSPs maintain their protein-lipid interactions after encapsulation. Fluorescence spectroscopy and quenching of the tryptophan residues with acrylamide, 5-DOXYL-stearic acid, and 16-DOXYL-stearic acid were used to determine the MSP orientation. We also utilized fluorescence anisotropy of tryptophans to measure the relative size of the NLPs and MSP aggregates after entrapment. Finally, circular dichroism spectroscopy was used to examine the secondary structure of the MSPs. Our results showed that, after entrapment, all of the lipid-bound MSPs maintained orientations that were minimally changed and indicative of association with lipids in NLPs. The tryptophan residues appeared to remain buried within the hydrophobic core of the lipid tails in the NLPs and appropriately spaced from the bilayer center. Also, after entrapment, lipid-bound MSPs maintained a high degree of α-helical content, a secondary structure associated with protein-lipid interactions. These findings demonstrate that NLPs are capable of serving as viable hosts for functional integral membrane proteins in the synthesis of sol-gel-derived bioinorganic hybrid nanomaterials.

Analysis of lipid phase behavior and protein conformational changes in nanolipoprotein particles upon entrapment in sol-gel-derived silica.

The entrapment of nanolipoprotein particles (NLPs) and liposomes in transparent, nanoporous silica gel derived from the precursor tetramethylorthosilicate was investigated. NLPs are discoidal patches of lipid bilayer that are belted by amphiphilic scaffold proteins and have an average thickness of 5 nm. The NLPs in this work had a diameter of roughly 15 nm and utilized membrane scaffold protein (MSP), a genetically altered variant of apolipoprotein A-I. Liposomes have previously been examined inside of silica sol-gels and have been shown to exhibit instability. This is attributed to their size (∼150 nm) and altered structure and constrained lipid dynamics upon entrapment within the nanometer-scale pores (5-50 nm) of the silica gel. By contrast, the dimensional match of NLPs with the intrinsic pore sizes of silica gel opens the possibility for their entrapment without disruption. Here we demonstrate that NLPs are more compatible with the nanometer-scale size of the porous environment by analysis of lipid phase behavior via fluorescence anisotropy and analysis of scaffold protein secondary structure via circular dichroism spectroscopy. Our results showed that the lipid phase behavior of NLPs entrapped inside of silica gel display closer resemblance to its solution behavior, more so than liposomes, and that the MSP in the NLPs maintain the high degree of α-helix secondary structure associated with functional protein-lipid interactions after entrapment. We also examined the effects of residual methanol on lipid phase behavior and the size of NLPs and found that it exerts different influences in solution and in silica gel; unlike in free solution, silica entrapment may be inhibiting NLP size increase and/or aggregation. These findings set precedence for a bioinorganic hybrid nanomaterial that could incorporate functional integral membrane proteins.