Discoidal High-density Lipoprotein Particles Research Articles

Lutein nanodisks protect human retinal pigment epithelial cells from UV light-induced damage.

The hydrophobic carotenoid, lutein, was conferred with aqueous solubility upon formulation into reconstituted discoidal high density lipoprotein particles, termed lutein nanodisks (ND). When formulated with phosphatidylcholine (PC), apolipoprotein (apo) A-I and lutein (formulation ratio = 5 mg PC/2 mg apoA-I/1 mg lutein), lutein solubilization efficiency in phosphate buffered saline (PBS) was ~90%. The UV/Vis absorbance maxima for lutein ND in PBS were red shifted by 6-13 nm versus the corresponding lutein absorbance maxima in ethanol. FPLC gel filtration chromatography gave rise to a single major absorbance peak in the size range of ND. Incubation of cultured ARPE-19 cells with lutein ND resulted in lutein uptake, as determined by HPLC analysis of cell extracts. Compared to control incubations, ARPE-19 cells incubated with lutein ND were protected from UV light-induced loss of cell viability. Consistent with this, reactive oxygen species generation, induced by exposure to UV irradiation, was lower in lutein-enriched cells than in control cells. Thus, uptake of ND-associated lutein protects ARPE-19 cells from UV light-induced damage. Taken together, the data indicate ND provide an aqueous lutein delivery vehicle for biotechnological or therapeutic applications.

Protein Backbone and Average Particle Dynamics in Reconstituted Discoidal and Spherical HDL Probed by Hydrogen Deuterium Exchange and Elastic Incoherent Neutron Scattering

Lipoproteins are supramolecular assemblies of proteins and lipids with dynamic characteristics critically linked to their biological functions as plasma lipid transporters and lipid exchangers. Among them, spherical high-density lipoproteins are the most abundant forms of high-density lipoprotein (HDL) in human plasma, active participants in reverse cholesterol transport, and associated with reduced development of atherosclerosis. Here, we employed elastic incoherent neutron scattering (EINS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the average particle dynamics and protein backbone local mobility of physiologically competent discoidal and spherical HDL particles reconstituted with human apolipoprotein A-I (apoA-I). Our EINS measurements indicated that discoidal HDL was more dynamic than spherical HDL at ambient temperatures, in agreement with their lipid-protein composition. Combining small-angle neutron scattering (SANS) with contrast variation and MS cross-linking, we showed earlier that the most likely organization of the three apolipoprotein A-I (apoA-I) chains in spherical HDL is a combination of a hairpin monomer and a helical antiparallel dimer. Here, we corroborated those findings with kinetic studies, employing hydrogen-deuterium exchange mass spectrometry (HDX-MS). Many overlapping apoA-I digested peptides exhibited bimodal HDX kinetics behavior, suggesting that apoA-I regions with the same amino acid composition located on different apoA-I chains had different conformations and/or interaction environments.

Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein

Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans.

Macrophages Shed Excess Cholesterol in Unique Extracellular Structures Containing Cholesterol Microdomains.

Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology. Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.

Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1–43 or 44–65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44–65 restored the immunoreactivity of mAbs whose epitope being within residues 44–65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles.

Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I.

High-density lipoprotein (HDL) particles are cholesterol and lipid transport containers. Mature HDL particles destined for the liver develop through the formation of intermediate discoidal HDL particles, which are the primary acceptors for cholesterol. Here we present the three-dimensional structure of reconstituted discoidal HDL (rdHDL) particles, using a shortened construct of human apolipoprotein A-I, determined from a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) data. The rdHDL particles feature a protein double belt surrounding a lipid bilayer patch in an antiparallel fashion. The integrity of this structure is maintained by up to 28 salt bridges and a zipper-like pattern of cation-π interactions between helices 4 and 6. To accommodate a hydrophobic interior, a gross 'right-to-right' rotation of the helices after lipidation is necessary. The structure reflects the complexity required for a shuttling container to hold a fluid lipid or cholesterol interior at a protein:lipid ratio of 1:50.

Cell lipid metabolism modulators 2-bromopalmitate, D609, monensin, U18666A and probucol shift discoidal HDL formation to the smaller-sized particles: implications for the mechanism of HDL assembly

ATP-binding cassette transporter A1 (ABCA1) mediates formation of disc-shaped high-density lipoprotein (HDL) from cell lipid and lipid-free apolipoprotein A-I (apo A-I). Discoidal HDL particles are heterogeneous in physicochemical characteristics for reasons that are understood incompletely. Discoidal lipoprotein particles similar in characteristics and heterogeneity to cell-formed discoidal HDL can be reconstituted from purified lipids and apo A-I by cell-free, physicochemical methods. The heterogeneity of reconstituted HDL (rHDL) is sensitive to the lipid composition of the starting lipid/apo A-I mixture. To determine whether the heterogeneity of cell-formed HDL is similarly sensitive to changes in cell lipids, we investigated four compounds that have well-established effects on cell lipid metabolism and ABCA1-mediated cell cholesterol efflux. 2-Bromopalmitate, D609, monensin and U18666A decreased formation of the larger-sized, but dramatically increased formation of the smaller-sized HDL. 2-Bromopalmitate did not appear to affect ABCA1 activity, subcellular localization or oligomerization, but induced dissolution of the cholesterol-phospholipid complexes in the plasma membrane. Arachidonic and linoleic acids shifted HDL formation to the smaller-sized species. Tangier disease mutations and inhibitors of ABCA1 activity wheat germ agglutinin and AG 490 reduced formation of both larger-sized and smaller-sized HDL. The effect of probucol was similar to the effect of 2-bromopalmitate. Taking rHDL formation as a paradigm, we propose that ABCA1 mutations and activity inhibitors reduce the amount of cell lipid available for HDL formation, and the compounds in the 2-bromopalmitate group and the polyunsaturated fatty acids change cell lipid composition from one that favors formation of the larger-sized HDL particles to one that favors formation of the smaller-sized species.

Kinetic and Thermodynamic Analyses of Spontaneous Exchange between High-Density Lipoprotein-Bound and Lipid-Free Apolipoprotein A-I

It is thought that apolipoprotein A-I (apoA-I) spontaneously exchanges between high-density lipoprotein (HDL)-bound and lipid-free states, which is relevant to the occurrence of preβ-HDL particles in plasma. To improve our understanding of the mechanistic basis for this phenomenon, we performed kinetic and thermodynamic analyses for apoA-I exchange between discoidal HDL-bound and lipid-free forms using fluorescence-labeled apoA-I variants. Gel filtration experiments demonstrated that addition of excess lipid-free apoA-I to discoidal HDL particles promotes exchange of apoA-I between HDL-associated and lipid-free pools without alteration of the steady-state HDL particle size. Kinetic analysis of time-dependent changes in NBD fluorescence upon the transition of NBD-labeled apoA-I from HDL-bound to lipid-free state indicates that the exchange kinetics are independent of the collision frequency between HDL-bound and lipid-free apoA-I, in which the lipid binding ability of apoA-I affects the rate of association of lipid-free apoA-I with the HDL particles and not the rate of dissociation of HDL-bound apoA-I. Thus, C-terminal truncations or mutations that reduce the lipid binding affinity of apoA-I strongly impair the transition of lipid-free apoA-I to the HDL-bound state. Thermodynamic analysis of the exchange kinetics demonstrated that the apoA-I exchange process is enthalpically unfavorable but entropically favorable. These results explain the thermodynamic basis of the spontaneous exchange reaction of apoA-I associated with HDL particles. The altered exchangeability of dysfunctional apoA-I would affect HDL particle rearrangement, leading to perturbed HDL metabolism.

Editorial: current research, knowledge and controversies on high density lipoprotein.

Editorial: current research, knowledge and controversies on high density lipoprotein.

Determination of the Dynamic Structures of Nacent Discoidal High-Density Lipoprotein (HDL) Bound to Lecithin Cholesterol Acyltransferase (LCAT) and Paraoxonase 1 (PON1)

The relative levels of plasma cholesterol in different lipoproteins are the major indicator for CVD and related diseases. Nascent discoidal HDL particles, which are then converted to spherical mature HDL by lecithin-cholesterol acyltransferase (LCAT), are integral in reverse cholesterol transport. Detailed knowledge of HDL structure is key to understanding its molecular mechanisms, including how Paraoxynase 1 (PON1) associates with the phospholipid surface of HDL helping prevent oxidation. Despite public interest in HDL structure related function, their highly dynamic nature has prevented precise structural determinations for nearly half a century. Here, we used our recently developed individual particle electron tomography (IPET) technique to study each individual complex of HDL-LCAT. The 3D reconstructions show, i) LCAT associates with HDL along a proposed mobile region of the apoA-1 backbone (res ∼122-142), where both monomers overlap antiparallel along the discoidal edge of HDL; ii) PON1 interacts with HDL through its hydrophobic tail, which binds into the phospholipid surface, and loosely associates with apoA-1. The structure provides novel information for modeling nascent discoidal HDL and further reveals the first view of HDL structural dynamics in three-dimensions. This result will benefit public knowledge and pharmaceutical drug design to control plasma HDL cholesterol level.

ABCA1 dimer–monomer interconversion during HDL generation revealed by single-molecule imaging

The generation of high-density lipoprotein (HDL), one of the most critical events for preventing atherosclerosis, is mediated by ATP-binding cassette protein A1 (ABCA1). ABCA1 is known to transfer cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I) for generating discoidal HDL (dHDL) particles, composed of 100-200 lipid molecules surrounded by two apoA-I molecules; however, the regulatory mechanisms are still poorly understood. Here we observed ABCA1-GFP and apoA-I at the level of single molecules on the plasma membrane via a total internal reflection fluorescence microscope. We found that about 70% of total ABCA1-GFP spots are immobilized on the plasma membrane and estimated that about 89% of immobile ABCA1 molecules are in dimers. Furthermore, an ATPase-deficient ABCA1 mutant failed to be immobilized or form a dimer. We found that the lipid acceptor apoA-I interacts with the ABCA1 dimer to generate dHDL and is followed by ABCA1 dimer-monomer interconversion. This indicates that the formation of the ABCA1 dimer is the key for apoA-I binding and nascent HDL generation. Our findings suggest the physiological significance of conversion of the ABCA1 monomer to a dimer: The dimer serves as a receptor for two apoA-I molecules for dHDL particle generation.

Stability of discoidal high-density lipoprotein particles

Motivated by experimental and numerical studies revealing that discoidal high-density lipoprotein (HDL) particles may adopt flat elliptical and nonplanar saddle-like configurations, it is hypothesized that these might represent stabilized configurations of initially unstable flat circular particles. A variational description is developed to explore the stability of a flat circular discoidal HDL particle. While the lipid bilayer is modeled as two-dimensional fluid film endowed with surface tension and bending elasticity, the apoA-I belt is modeled as one-dimensional inextensible twist-free chain endowed with bending elasticity. Stability is investigated using the second variation of the underlying energy functional. Various planar and nonplanar instability modes are predicted and corresponding nondimensional critical values of salient dimensionless parameters are obtained. The results predict that the first planar and nonplanar unstable modes occur due to in-plane elliptical and transverse saddle-like perturbations. Based on available data, detailed stability diagrams indicate the range of input parameters for which a flat circular discoidal HDL particle is linearly stable or unstable.

Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6nm) and small (7.8nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5kcal/mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.

Apolipoprotein A-II-mediated Conformational Changes of Apolipoprotein A-I in Discoidal High Density Lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.

Crystal Structure of C-terminal Truncated Apolipoprotein A-I Reveals the Assembly of High Density Lipoprotein (HDL) by Dimerization

Apolipoprotein A-I (apoA-I) plays important structural and functional roles in plasma high density lipoprotein (HDL) that is responsible for reverse cholesterol transport. However, a molecular understanding of HDL assembly and function remains enigmatic. The 2.2-Å crystal structure of Δ(185-243)apoA-I reported here shows that it forms a half-circle dimer. The backbone of the dimer consists of two elongated antiparallel proline-kinked helices (five AB tandem repeats). The N-terminal domain of each molecule forms a four-helix bundle with the helical C-terminal region of the symmetry-related partner. The central region forms a flexible domain with two antiparallel helices connecting the bundles at each end. The two-domain dimer structure based on helical repeats suggests the role of apoA-I in the formation of discoidal HDL particles. Furthermore, the structure suggests the possible interaction with lecithin-cholesterol acyltransferase and may shed light on the molecular details of the effect of the Milano, Paris, and Fin mutations.

Domains of apoE4 required for the biogenesis of apoE-containing HDL

Introduction. We have studied the functions of truncated apoE4 forms in vitro and in vivo in order to identify the domains of apoE4 required for the biogenesis of apoE-containing high-density lipoprotein (HDL). Results. We have found that apoE4-185, -202, -229, or -259 could promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux in vitro, although less efficiently than Full-length apoE4, and had diminished capacity to activate lecithin cholesterol acyltransferase (LCAT). Formation of HDL in vivo was assessed by various methods following gene transfer in apolipoprotein A-I−/− × apoE−/− mice. Fast protein liquid chromatography of plasma showed that the truncated apoE forms, except apoE4-185, generated an apoE-containing HDL peak. Two-dimensional gel electrophoresis of plasma and electron microscopy showed that truncated apoE forms generated distinct HDL subpopulations and formed discoidal HDL particles which could be converted to spherical by co-administration of truncated apoE4-202 and LCAT. Conclusion. Overall, the in-vivo and in-vitro data are consistent and indicate that apoE4-185 is the shortest truncated form that supports formation of discoidal apoE4-containing HDL particles.

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190-243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.

SEIRA Spectroscopy on a Membrane Receptor Monolayer Using Lipoprotein Particles as Carriers

Surface-enhanced infrared absorption (SEIRA) difference spectroscopy can probe reactions in a protein monolayer tethered to a nanostructured gold surface. SEIRA studies of membrane proteins, however, remain challenging due to sample stability, effects of the metal surface on function, and the need for a membrane-mimicking environment. Here we demonstrate and characterize a model system for membrane receptor investigations using SEIRA spectroscopy. The system employs nanoscale apolipoprotein bound bilayer (NABB) particles, similar to discoidal high-density lipoprotein particles, as soluble carriers for the G-protein-coupled receptor rhodopsin. The His-tag of the engineered apolipoprotein allows for selective binding of the NABBs to a Ni-NTA modified surface, while the lipid environment of the particle ensures stability and protection of the embedded receptor. Using SEIRA spectroscopy, we followed specific binding of rhodopsin-loaded NABB particles to the surface and formation of a membrane protein monolayer. Functionality of the photoreceptor in the immobilized NABBs was probed by SEIRA difference spectroscopy confirming protein conformational changes associated with photoactivation. Orientation of the immobilized NABB particles was assessed by comparing SEIRA data with polarized attenuated total reflection-Fourier-transform infrared spectroscopy. Thus, SEIRA difference spectroscopy supported by the NABB technology provides a promising approach for further functional studies of transmembrane receptors.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Assembly of Lipids and Proteins into Lipoprotein Particles

The self-assembly of reconstituted discoidal high-density lipoproteins, known as nanodiscs, was studied using coarse-grained molecular dynamics and small-angle X-ray scattering. In humans, high-density lipoprotein particles transport cholesterol in the blood and facilitate the removal of excess cholesterol from the body. Native high-density lipoprotein exhibits a wide variety of shapes and sizes, forming lipid-free/poor, nascent discoidal, and mature spherical particles. Little is known about how these lipoprotein particles assemble and transform from one state to another. Multiple 10 micros coarse-grained simulations reveal the assembly of discoidal high-density lipoprotein particles from disordered protein-lipid complexes. Small-angle X-ray scattering patterns were calculated from the final assembled structures and compared with experimental measurements carried out for this study to verify the accuracy of the coarse-grained simulations. Results show that hydrophobic interactions assemble, within several microseconds, the amphipathic helical proteins and lipids into roughly discoidal particles, while the proteins assume a final approximate double-belt configuration on a slower time scale.