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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.

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.

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.

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.

One of the hallmarks of Alzheimer's disease (AD) pathogenesis is believed to be the production and deposition of amyloid-beta (Aβ) peptide into extracellular plaques. Existing research indicates that extracellular vesicles (EVs) can carry Aβ associated with AD. However, characterization of the EVs-associated Aβ 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 Aβ 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 Aβ 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 Aβ 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 Aβ 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 Aβ 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., Extracellular vesicles can carry Aβ associated with Alzheimer's disease. In this article we identify specific Raman spectroscopic features that can be associated with Aβ present in the molecular cargo of small extracellular vesicles.

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.

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.

The soluble oligomeric form of the amyloid beta (Aβ) 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 Aβ. 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 Aβ and measurements of layer thickness alterations inform on the mechanism underlying the ability of SLF to inhibit Aβ toxicity and its progression towards larger oligomeric assemblies. Depending on the oligomeric state of Aβ, distinct binding affinities for SLF are revealed. The Aβ monomer and dimer uniquely possess sub-nanomolar affinity for SLF via a non-specific mode of binding. SLF binding is weaker in oligomeric Aβ, 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 Aβ peptide. The MP-SPR results together with in silico modeling provide affinity data for the SLF-Aβ interaction and allow us to develop a new general method for examining protein aggregation.

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.

Soluble, small amyloid- β oligomers ( A β O ) 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- β ( A β ) 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 A β peptides and spin-labeled fluorine (SLF), a peptide aggregation inhibitor. Changes in the peak positions and intensity ratios of two spectral peaks near 1600 c m − 1 and 2900 c m − 1 can be used to monitor the molecular interactions between SLF and A β . This study demonstrates the potential of SERS spectroscopy for rapidly screening and identifying new anti- A β therapeutic agents.

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.

There is growing recognition regarding the role of intracellular amyloid beta (A&beta, ) in the Alzheimer&rsquo, s disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal A&beta, 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 A&beta, 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 A&beta, 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 A&beta, Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular A&beta, Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular A&beta, 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 A&beta, 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 A&beta, its promotion of ROS, and A&beta, metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and A&beta, oligomer formation.

Proteins predicted to be composed of large stretches of coiled-coil structure have often proven difficult to crystallize for structural determination. We have successfully applied EPR spectroscopic techniques to the study of the structure and assembly of full-length human vimentin assembled into native 11 nm filaments, in physiologic solution, circumventing the limitations of crystallizing shorter peptide sequences. Tektins are a small family of highly alpha helical filamentous proteins found in the doublet microtubules of cilia and related structures. Tektins exhibit several similarities to intermediate filaments (IFs): moderate molecular weight, highly alpha helical, hypothesized to be coiled-coil, and homo- and heteromeric assembly into long smooth filaments. In this report, we show the application of IF research methodologies to the study of tektin structure and assembly. To begin in vitro studies, expression constructs for human tektins 1, 2, and 4 were synthesized. Recombinant tektins were produced in E. coli and purified by chromatography. Preparations of tektin 1 successfully formed filaments. The recombinant human tektin 1 was used to produce antibodies which recognized an antigen in mouse testes, most likely present in sperm flagella. Finally, we report the creation of seven mutants to analyze predictions of coiled-coil structure in the rod 1A domain of tektin 1. Although this region is predicted to be coiled-coil, our EPR analysis does not reflect the parallel, in register, coiled-coil structure as demonstrated in vimentin and kinesin. These results document that tektin can be successfully expressed and assembled in vitro, and that SDSL EPR techniques can be used for structural analysis.

Amyloid-β (Aβ) protein causes neurotoxicity and its abnormal aggregation into amyloid is a pathological hallmark of Alzheimer's disease (AD). Cellular proteins able to interact with Aβ or its precursor, AβPP (amyloid-β protein precursor), may regulate Aβ production and neurotoxicity. We identified a brain-enriched type I transmembrane protein, tomoregulin (TR), that directly binds Aβ and Aβ oligomers (AβO). TR co-immunoprecipitated with Aβ and AβO in cultured cells and co-localized with amyloid plaques and intraneuronal Aβ in the 5xFAD AD mouse model. TR was also enriched in astrocytic processes reactive to amyloid plaques. Surface plasmon resonance spectroscopy studies showed that the extracellular domain of TR binds to AβO with a high affinity (KD = 76.8 nM). Electron paramagnetic resonance spectroscopy also demonstrated a physical interaction between spin-labeled Aβ and the TR extracellular domain in solution. Furthermore, TR also interacted with AβPP and enhanced its cleavage by α-secretase. Both cellular expression of TR and application of recombinant TR extracellular domain protected N2a neurons from AβO-induced neuronal death. These data provide first evidence that neuronal and astrocytic expression of TR is intimately related to Aβ metabolism and toxicity, and could be neuroprotective through its direct interaction with Aβ andAβPP.

The identification of environmental factors that lead to loss of tolerance has been coined the holy grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein, we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177-314 of PDC-E2 by joining a recombinant peptide spanning residues 177-252 (PDC-228) with a 62-residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 inner lipoyl domain (ILD). We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA-modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the immunoglobulin M isotype and more dominant in early-stage primary biliary cholangitis (PBC), suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition, we analyzed PPL, LA-PPL, and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by enzyme-linked immunosorbent assay, immunoblotting, and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid. Conclusion: A molecular understanding of the conformation of xenobiotic-modified PDC-E2 is critical for understanding xenobiotic modification and loss of tolerance in PBC with widespread implications for a role of environmental chemicals in the induction of autoimmunity. (Hepatology 2017;65:1670-1682).

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.

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²⁺-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²⁺-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²⁺-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²⁺-free recoverin and 3.7 ± 0.1 nm for Ca²⁺-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²⁺-bound recoverin may represent an intermolecular distance between C39 and N120. ¹⁵N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²⁺-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca²⁺-free recoverin is monomeric. We propose that Ca²⁺-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²⁺-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²⁺-induced dimerization is functionally important but not well understood.

In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na+/Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate.

Apolipoprotein A-I (ApoA-I) is the major protein component of high-density lipoprotein (HDL), and is critical for maintenance of cholesterol homeostasis. During reverse cholesterol transport, HDL transitions between an array of subclasses, differing in size and composition. This process requires ApoA-I to adapt to changes in the shape of the HDL particle, transiting from an apolipoprotein to a myriad of HDL subclass-specific conformations. Changes in ApoA-I structure cause alterations in HDL-specific enzyme and receptor-binding properties, and thereby direct the HDL particle through the reverse cholesterol transport pathway. In this study, we used site-directed spin label spectroscopy to examine the conformational details of the ApoA-I central domain on HDL. The motional dynamics and accessibility to hydrophobic/hydrophilic relaxation agents of ApoA-I residues 99–163 on 9.6-nm reconstituted HDL was analyzed by EPR. In previous analyses, we examined residues 6–98 and 164–238 (of ApoA-I's 243 residues), and combining these findings with the current results, we have generated a full-length map of the backbone structure of reconstituted HDL-associated ApoA-I. Remarkably, given that the majority of ApoA-I's length is composed of amphipathic helices, we have identified nonhelical residues, specifically the presence of a β-strand (residues 149–157). The significance of these nonhelical residues is discussed, along with the other features, in the context of ApoA-I function in contrast to recent models derived by other methods.

We have engineered apolipoprotein A-I (apoA-I), a major protein constituent of high-density lipoprotein (HDL), to contain DOTA-chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism and regulation in vivo. This protein contrast agent was obtained by attaching the thiol-reactive Gd[MTS-ADO3A] label at Cys residues replaced at four distinct positions (52, 55, 76 and 80) in apoA-I. MRI of infused mice previously showed that the Gd-labeled apoA-I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA-I are superior when the ADO3A moiety is located at position 55, compared with the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X-band (9 GHz) electron paramagnetic resonance (EPR) spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid-free with the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid-free and rHDL states correlate to the product of the X-band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL-associated apoA-I containing the Gd[MTS-ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X-band EPR. While room temperature EPR requires >1 m m Gd(III)-labeled and only >10 µ m nitroxide-labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5 µl). Thus, X-band EPR provides a practical assessment for the suitability of imaging candidates containing the site-directed ADO3A contrast probe.

Very little data have been reported that describe the structure of the tail domain of any cytoplasmic intermediate filament (IF) protein. We report here the results of studies using site directed spin labeling and electron paramagnetic resonance (SDSL-EPR) to explore the structure and dynamics of the tail domain of human vimentin in tetramers (protofilaments) and filaments. The data demonstrate that in contrast to the vimentin head and rod domains, the tail domains are not closely apposed in protofilaments. However, upon assembly into intact IFs, several sites, including positions 445, 446, 451, and 452, the conserved “beta-site,” become closely apposed, indicating dynamic changes in tail domain structure that accompany filament elongation. No evidence is seen for coiled-coil structure within the region studied, in either protofilaments or assembled filaments. EPR analysis also establishes that more than half of the tail domain is very flexible in both the assembly intermediate and the intact IF. However, by positioning the spin label at distinct sites, EPR is able to identify both the rod proximal region and sites flanking the beta-site motif as rigid locations within the tail. The rod proximal region is well assembled at the tetramer stage with only slight changes occurring during filament elongation. In contrast, at the beta site, the polypeptide backbone transitions from flexible in the assembly intermediate to much more rigid in the intact IF. These data support a model in which the distal tail domain structure undergoes significant conformational change during filament elongation and final assembly.

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.

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.

Rats with nephrotic syndrome (NS) have a fivefold increase in lipids and a similar decrease in triglyceride-rich lipoprotein (TRL) clearance. Lipoprotein lipase (LPL) is reduced both in NS and in the Nagase analbuminemic rat. These rats have nearly normal triglyceride levels and TRL clearance, suggesting that reduction in LPL alone is insufficient to cause increased TRL levels. Apolipoprotein E (apoE) was decreased in lipoprotein fractions in NS, but not in analbuminemia. Here we tested whether decreased apoE binding to lipoproteins in NS contributes to hyperlipidemia by decreasing their affinity for lipoprotein receptors. Plasma apoE was increased 60% in both NS and analbuminemia compared with control (CTRL) as a result of a 60% decreased apoE clearance. Very-low-density lipoprotein and high-density lipoprotein in NS had significantly less apoE per mole of phospholipid compared with analbuminemia or CTRL and significantly greater lipid content; however, apoE binding did not differ by lipoprotein class or group. There was a significant reduction of receptors for lipoproteins in nearly all tissues in NS compared with CTRL and analbuminemia. Thus, apoE within lipoprotein fractions was reduced by dilution resulting from expansion of the lipid fraction due to decreased lipolysis and not to differing affinity for apoE. Decreased lipoprotein receptors result from proteinuria and contribute to hyperlipidemia in NS.

The field of medicinal chemistry applied to the discovery of novel and efficacious therapy for neurodegenerative diseases is ever changing as new molecular targets, compounds and technologies are discovered. Isaac Bruce, Commissioning Editor, spoke to six experts and discussed why they became involved in the field, the future of the area and the challenges it faces.

Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.

Apolipoprotein A-I plays a central role in the solution structure of high-density lipoproteins. Determining the stoichiometry of lipid-bound apo A-I in the hydrated state is therefore fundamental to understanding how high-density lipoproteins form and function. Here, we use the quantum optical phenomenon of photon antibunching to determine the number of apo A-I molecules bound to discoidal lipoproteins and compare this with values obtained by photon-counting histogram analysis. Both the photon antibunching and photon-counting analyses show that reconstituted high-density lipoprotein particles contain two apo A-I molecules, which is in agreement with the commonly accepted double-belt model.

Coronary disease risk increases inversely with high-density lipoprotein (HDL) level. The measurement of the biodistribution and clearance of HDL in vivo, however, has posed a technical challenge. This study presents an approach to the development of a lipoprotein MRI agent by linking gadolinium methanethiosulfonate (Gd[MTS-ADO3A]) to a selective cysteine mutation in position 55 of apo AI, the major protein of HDL. The contrast agent targets both liver and kidney, the sites of HDL catabolism, whereas the standard MRI contrast agent, gadolinium-diethylenetriaminepentaacetic acid-bismethylamide (GdDTPA-BMA, gadodiamide), enhances only the kidney image. Using a modified apolipoprotein AI to create an HDL contrast agent provides a new approach to investigate HDL biodistribution, metabolism and regulation in vivo.

The molecular pathogenesis of disorders arising from protein mis-folding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex mis-folding and aggregation that initiates the toxic cascade resulting in Alzheimer's disease, we have developed a TOAC spin-labeled Aβ peptide to observe its isoform-dependent interaction with the apoE protein. While most individuals carry the E3 isoform of apoE, approximately 15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimer's. ApoE is consistently associated with the amyloid plaque marker for Alzheimer's disease. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aβ peptide processing and hence Aβ toxicity. We employed EPR spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aβ in solution. EPR spectra of the spin labeled side chain report on side chain and backbone dynamics, as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C-terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aβ oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimer's risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aβ.

The ability to exogenously present cell-surface receptors in high-affinity conformations in a synthetic system offers an opportunity to provide host cells with protection from pathogenic toxins. This strategy requires improvement of the synthetic receptor binding affinity against its native counterpart, particularly with polyvalent toxins where clustering of membrane receptors can hinder binding. Here we demonstrate that reconstituted lipoprotein, nanometer-sized discoidal lipid bilayers bounded by apolipoprotein and functionalized by incorporation of pathogen receptors, provides a means to enhance toxin-receptor binding through molecular-level control over the receptor microenvironment (specifically, its rigidity, composition, and heterogeneity). Using a Foerster Resonance Energy Transfer (FRET)-based assay, we found that reconstituted lipoprotein incorporating low concentrations of ganglioside monosialotetrahexosylganglioside (GM1) binds polymeric cholera toxin with significantly higher affinity than liposomes or supported lipid bilayers, most likely a result of the enhanced control over receptor clustering provided by the lipoprotein platform. Using wide-area epifluorescence, we found that this enhanced binding capacity can be effectively utilized to divert cholera toxin away from populations of healthy mammalian cells. In summary, we found that reconstitutions of high-density lipoprotein can be engineered to include specific pathogen receptors; that their pathogen binding affinity is altered, presumably due to attenuation of receptor aggregation; and that these assemblies are effective at protecting cells from biological toxins.

Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL approximately LDL

We have used site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to identify residues 17 and 137 as sites of interaction between the head domain and rod domain 1A of the intermediate filament protein vimentin. This interaction was maximal when compared with the spin labels placed at up- and downstream positions in both head and rod regions, indicating that residues 17 and 137 were the closest point of interaction in this region. SDSL EPR characterization of residues 120-145, which includes the site of head contact with rod 1A, reveals that this region exhibits the heptad repeat pattern indicative of α-helical coiled-coil structure, but that this heptad repeat pattern begins to decay near residue 139, suggesting a transition out of coiled-coil structure. By monitoring the spectra of spin labels placed at the 17 and 137 residues during in vitro assembly, we show that 17-137 interaction occurs early in the assembly process. We also explored the effect of phosphorylation on the 17-137 interaction and found that phosphorylation-induced changes affected the head-head interaction (17-17) in the dimer, without significantly influencing the rod-rod (137-137) and head-rod (17-137) interactions in the dimer. These data provide the first direct evidence for, and location of, headrod interactions in assembled intermediate filaments, as well as direct evidence of coiled-coil structure in rod 1A. Finally, the data identify changes in the structure in this region following in vitro phosphorylation.

We studied the reaction between hypochlorite anion ( − OCl) and hydrogen peroxide (H 2 O 2 ) by using spin-trapping agents such as 5,5-dimethyl-1-pyrroline- N -oxide (DMPO) and 5-diethoxy-phosphoryl-5-methyl-1-pyrroline- N -oxide (DEPMPO). The obtained data demonstrate that hydroxyl radical is produced in this reaction. Thus, − OCl and H 2 O 2 could assume new relevance in environmental chemistry by representing a potential source of hydroxyl radicals and other oxidant species, such as HO–OCl and OCl radical.

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.

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.

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

© 2015 American Chemical Society. 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.

Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to α helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.

Intermediate filaments (IFs1) are one of the three major classes of cytoskeletal structures in metazoan cells. Though more than 60 IF genes have been defined in the human genome, most IFs are assembled from just 1 or 2 IF proteins. Which specific IF protein(s) is/are expressed varies by cell type and the state of development/differentiation (1–4). Though there is considerable diversity in the IF family in both size and primary sequence, the vast majority of IF proteins show the conservation of several features: (1) they are localized to and/or can assemble into 8–11 nm IFs, and (2) they exhibit a common predicted domain structure. The predicted domain structure consists of (a) head and tail domains that vary widely in size and primary sequence and show no obvious predicted secondary structure and (b) a central rod domain that is approximately 310 amino acids in length. This rod domain is predicted to consist of extended runs of an α helix containing a heptad repeat pattern, which predicts the formation of coiled-coils (coil domains) separated by short regions called linkers that are not predicted to be α-helical, nor do they show a heptad repeat pattern (1–3, 5–16). However, depending on how parameters are set in programs that make such predictions, the strength of the coiled-coil prediction can vary quite dramatically (Figure 1) (17). Importantly, no direct test of structure has ever been made in these linker regions. FIGURE 1 Vimentin Schematic. A traditional schematic of the vimentin molecule is shown, with coil domains flanking the noncoil or linker region. Three variants of Coils version 2.1 (http://www.ch.embnet.org/software/COILS) predictions used to generate such models ... No intact IF protein or IF has been crystallized, thus a high-resolution structural map of neither the IF nor the IF protein has been determined. However, a variety of approaches, both theoretical and experimental have contributed to a model of IF structure and assembly. Strelkov and co-workers, for example, have been successful at the crystallization of fragments of the homopolymeric protein vimentin (18, 19). From the solution of these crystals’ structures has come the first detailed pictures of 2 regions of IF coiled-coil structure. The higher order structure of IF proteins within a filament and within assembly intermediates has been approached with cross-linking methodology. From both vimentin and keratin cross-link studies have emerged data that suggest a set of overlapping structures that are found in assembled filaments (20–23). We have employed site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to characterize the full range of IF structure, yielding data on secondary, tertiary, and quaternary structures, and identifying sites where proteins are in close contact with one another in intact filaments (24–26). Collectively, these data support a model of the IF as beginning with the formation of an in-parallel and in-register dimer, where the central rod domain forms, at least in part, a coiled-coil dimer. Dimers then assemble into antiparallel tetramers with rod domains 1 associated (A11 interaction), followed by a species with rod domains 2 associated in an antiparallel interaction (A22 interaction). Cross-linking and SDSL–EPR have both contributed to establishing relationships between adjacent dimers, providing data on the relative orientation and registry of adjacent dimers. Most data that have been generated address the structure of the rod domain and its assembly into a coiled-coil dimer, and the relationship between adjacent rod domains in higher order structures. Essentially, nothing is known about the structure of the linker domains, and very little data has been generated that addresses the structure of either head or tail domains. In this article, we use SDSL-EPR to describe a series of spin-labeled mutants in the linker region of human vimentin (residues 281–304). EPR analysis of these spin-labeled mutants indicates that this region lacks coiled-coil structure, but also shows that several sites in this linker region are positioned extremely close to one another in intact filaments and that the polypeptide backbone in this region is quite rigid. Of considerable interest is evidence that the region from 283–291 assembles into its final structure much earlier than the coiled-coil domains within rod 1B and 2B that we have examined and is highly resistant to thermal denaturation.

Interaction of the amino- and carboxyl-terminal domains in apolipoprotein (apo) E, referred to as domain interaction, is predicted to be more pronounced in apoE4 than in apoE3 and to underlie the association of apoE4 with Alzheimer and cardiovascular diseases. However, direct physical proof for the domain interaction concept is lacking. To address this issue, fluorescence resonance energy transfer and electron paramagnetic resonance spectroscopy were used to probe the spatial proximity of the two domains of apoE. Both methods demonstrated that the two domains are closer in both lipid-free and phospholipid-bound apoE4 than in apoE3 as a result of domain interaction. In addition, as shown by electron paramagnetic resonance, the domains of apoE4 move apart to resemble more closely the distance in apoE3 when the isoforms are bound to triglyceride-rich emulsion particles. These results demonstrate that domain interaction is a structural property of apoE4 and that apoE adopts different conformations when complexed to different lipids.