Apolipoprotein A-I is the major protein in high-density lipoprotein (HDL) and plays an important ... more Apolipoprotein A-I is the major protein in high-density lipoprotein (HDL) and plays an important role during the process of reverse cholesterol transport (RCT). Knowledge of the high-resolution structure of full-length apoA-I is vital for a molecular understanding of the function of HDL at the various steps of the RCT pathway. Due to the flexible nature of apoA-I and aggregation properties, the structure of full-length lipid-free apoA-I has evaded description for over three decades. Sequence analysis of apoA-I suggested that the amphipathic α-helix is the structural motif of exchangeable apolipoprotein, and NMR, X-ray and MD simulation studies have confirmed this. Different laboratories have used different methods to probe the secondary structure distribution and organization of both the lipid-free and lipid-bound apoA-I structure. Mutation analysis, synthetic peptide models, surface chemistry and crystal structures have converged on the lipid-free apoA-I domain structure and functi...
We found earlier that apoA-I variants that induced hypertriglyceridemia (HTG) in mice had increas... more We found earlier that apoA-I variants that induced hypertriglyceridemia (HTG) in mice had increased affinity to TG-rich lipoproteins and thereby impaired their catabolism. Here, we tested whether a naturally occurring human apoA-I mutation, Lys107del, associated with HTG also promotes apoA-I binding to TG-rich particles. We expressed apoA-I[Lys107del] variant in Escherichia coli, studied its binding to TG-rich emulsion particles, and performed a physicochemical characterization of the protein. Compared with WT apoA-I, apoA-I[Lys107del] showed enhanced binding to TG-rich particles, lower stability, and greater exposure of hydrophobic surfaces. The crystal structure of truncated, Δ(185-243), apoA-I suggests that deletion of Lys107 disrupts helix registration and disturbs a stabilizing salt bridge network in the N-terminal helical bundle. To elucidate the structural changes responsible for the altered function of apoA-I[Lys107del], we studied another mutant, apoA-I [Lys107Ala]. Our fin...
CONTENTS I. INTRODUCTION II. CHEMICAL FORMULATION AND NOMENCLATURE III. PHYSICAL STATES OF CHOLES... more CONTENTS I. INTRODUCTION II. CHEMICAL FORMULATION AND NOMENCLATURE III. PHYSICAL STATES OF CHOLESTERYL ESTERS A. Crystalline states 1. Monolayer type I 2. Monolayer type II 3. Bilayer B. The mesomorphic state C. The liquid state IV. TRANSITION BETWEEN STATES V. PHASE BEHAVIOR OF CHOLESTERYL ESTERS FROM POLARIZING MICROSCOPY AND DIFFERENTIAL SCANNING CALORIMETRY A. Effects of chain length in saturated and unsaturated cholesteryl esters B. Influence of the unsaturation in the fatty acyl chain 1. Effect of double bond position 2. Effect of increasing unsaturation VISCOSITY, FLUIDITY AND MOLECULAR MOTIONS CRYSTAL AND SMECTIC MESOPHASE STRUCTURE OF CHOLESTERYL
Proceedings of the National Academy of Sciences, 1996
Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in th... more Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximately 7, by using differential scanning calorimetry and circular dichroism. At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint Tm 57 +/- 1 degree C, calorimetric enthalpy deltaH(Tm)= 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy deltaHv(Tm) approximately 32.5 kcal/mol, and cooperativity deltaHv(Tm)/deltaH(Tm) approximately 0.16. The enthalpy deltaH(Tm) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximately 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal non...
properties of apoA-I arise primarily through its important roles in the pathway of reverse choles... more properties of apoA-I arise primarily through its important roles in the pathway of reverse cholesterol transport; where apoA-I stabilizes and maintains the structure of HDL particles, promotes cellular cholesterol effl ux by binding to specifi c ATP binding cassette transporters, interacts with the enzyme lecithin:cholesterol acyltransferase (LCAT) to drive the maturation of HDL particles, binds and modifi es the lipoprotein surface to facilitate enzyme reactions, and interacts with the scavenger receptor class B type 1 for selective cholesterol ester (CE) uptake by the liver for excretion (2-4). ApoA-I exists in lipid-free, lipid-poor, and lipid-bound states in plasma, and exchanges among HDL and TAGrich lipoprotein particles like very low density lipoproteins (VLDLs) and chylomicrons (CMs) (5). ApoA-I interacts primarily with the hydrocarbon chains of the phospholipids (PL) on discoidal and spherical HDLs (6-8) and may also interact with the hydrophobic CE core of spherical HDL and the hydrophobic TAG core of TAG-rich lipoproteins. The conformational fl exibility of apoA-I allows it to adopt a variety of conformations involving lipid adsorption, partial or full desorption, and fl exible unfolding and refolding in diverse physical environments to facilitate its multiple functions. Studies of the molecular mechanisms of the lipid association and the conformational fl exibility of apoA-I are essential for understanding the structure-function relationships. ApoA-I has an exon 3 encoded region (residues 1-43), and an exon 4 encoded region (residues 44-243) that contains 10 11/22-mer tandem repeat amino acid segments that are predicted to form class A and class Y amphipathic ␣-helices (A ␣ Hs) (9, 10). These A ␣ Hs (helix 1-10) are the lipid binding motif of apoA-I and the structural basis for its multiple functions. Segment deletion and point mutation studies have elucidated the possible conformation and functions for each helical segment (11-16). Several studies have suggested that the lipid-free apoA-I molecule possesses a Abstract Apolipoprotein A-I (apoA-I) has a great conformational fl exibility to exist in lipid-free, lipid-poor, and lipid-bound states during lipid metabolism. To address the lipid binding and the dynamic desorption behavior of apoA-I at lipoprotein surfaces, apoA-I, ⌬ (185-243)apoA-I, and ⌬ (1-59)(185-243)apoA-I were studied at triolein/water and phosphatidylcholine/triolein/water interfaces with special attention to surface pressure. All three proteins are surface active to both interfaces lowering the interfacial tension and thus increasing the surface pressure to modify the interfaces. ⌬ (185-243)apoA-I adsorbs much more slowly and lowers the interfacial tension less than full-length apoA-I, confi rming that the C-terminal domain (residues 185-243) initiates the lipid binding. ⌬ (1-59)(185-243)apoA-I binds more rapidly and lowers the interfacial tension more than ⌬ (185-243)apoA-I, suggesting that destabilizing the N-terminal ␣-helical bundle (residues 1-185) restores lipid binding. The three proteins desorb from both interfaces at different surface pressures revealing that different domains of apoA-I possess different lipid affi nity. ⌬ (1-59)(185-243)apoA-I desorbs at lower pressures compared with apoA-I and ⌬ (185-243) apoA-I indicating that it is missing a strong lipid association motif. We propose that during lipoprotein remodeling, surface pressure mediates the adsorption and partial or full desorption of apoA-I allowing it to exchange among different lipoproteins and adopt various conformations to facilitate its multiple functions.
that raise HDL levels do not necessarily confer additional cardioprotection (Refs. 1, 2 and refer... more that raise HDL levels do not necessarily confer additional cardioprotection (Refs. 1, 2 and references therein). Plasma HDL carries from two to fi ve copies of apoA-I depending on the particle size (8-12 nm) and varies in particle shape (nascent "discoidal" or mature "spherical", Fig. 1), protein conformation, biochemical composition, and functional properties (1). The nascent HDL particle is generally envisioned as a contorted disc composed of a cholesterolcontaining phospholipid bilayer that is encircled by two antiparallel apoA-I molecules in a highly ␣-helical dynamic "double-belt" conformation (3-5). Micelle-like lipid packing has also been proposed in some studies (Ref. 6 and references therein). ApoA-I on HDL activates LCAT that converts nascent HDL into mature spherical particles containing a core of apolar lipids, mainly cholesterol esters, and a small amount of triacylglycerides. ApoA-I on mature HDL is proposed to form a double belt similar to that on nascent HDL but bent in a "trefoil/tetrafoil" fashion to confer 2D curvature to the surface of spherical particles (7, 8) (Fig. 1B). ApoA-I on the HDL surface forms a fl exible structural scaffold and is an important ligand for many functional interactions that direct HDL metabolism (1, 7-11). The amino acid sequence of apoA-I starts with a G-repeat (residues 1-43) followed by ten Pro-punctuated tandem Abstract HDL removes cell cholesterol and protects against atherosclerosis. ApoA-I provides a fl exible structural scaffold and an important functional ligand on the HDL surface. We propose structural models for apoA-I Milano (R173C) and apoA-I Paris (R151C) mutants that show high cardioprotection despite low HDL levels. Previous studies established that two apoA-I molecules encircle HDL in an antiparallel, helical double-belt conformation. Recently, we solved the atomic structure of lipid-free ⌬ (185-243)apoA-I and proposed a conformational ensemble for apoA-I WT on HDL. Here we modify this ensemble to understand how intermolecular disulfi des involving C173 or C151 infl uence protein conformation. The double-belt conformations are modifi ed by belt rotation, main-chain unhinging around Gly, and Pro-induced helical bending, and they are verifi ed by comparison with previous experimental studies and by molecular dynamics simulations of apoA-I Milano homodimer. In our models, the molecular termini repack on various-sized HDL, while packing around helix-5 in apoA-I WT , helix-6 in apoA-I Paris , or helix-7 in apoA-I Milano homodimer is largely conserved. We propose how the disulfi de-induced constraints alter the protein conformation and facilitate dissociation of the C-terminal segment from HDL to recruit additional lipid. Our models unify previous studies of apoA-I Milano and demonstrate how the mutational effects propagate to the molecular termini, altering their conformations, dynamics, and function.-Gursky, O.
The primary and secondary structure of human plasma apolipoprotein A-I and apolipoprotein E-3 hav... more The primary and secondary structure of human plasma apolipoprotein A-I and apolipoprotein E-3 have been analyzed to further our understanding of the secondary and tertiary conformation of these proteins and the structure and function of plasma lipoprotein particles. The methods used to analyze the primary sequence of these proteins used computer programs: (a) to identify repeated patterns within these proteins on the basis of conservative substitutions and similarities within the physicochemical properties of each residue; (b) for local averaging, hydrophobic moment, and Fourier analysis of the physicochemical properties; and (c) for secondary structure prediction of each protein carried out using homology, statistical, and information theory based methods. Circular dichroism was used to study purified lipid-protein complexes of each protein and quantitate the secondary structure in a lipid environment. The data from these analyses were integrated into a single secondary structure prediction to derive a model of each protein. The sequence homology within apolipoproteins A-I, E-3, and A-IV is used to derive a consensus sequence for two 11 amino acid repeating sequences in this family of proteins.
Apolipoprotein A-I (apoA-I) is the main protein of plasma high-density lipoproteins (HDL, or good... more Apolipoprotein A-I (apoA-I) is the main protein of plasma high-density lipoproteins (HDL, or good cholesterol) that remove excess cell cholesterol and protect against atherosclerosis. In hereditary amyloidosis, mutations in apoA-I promote its proteolysis and the deposition of the 9-11 kDa N-terminal fragments as fibrils in vital organs such as kidney, liver, and heart, causing organ damage. All known amyloidogenic mutations in human apoA-I are clustered in two residue segments, 26-107 and 154-178. The X-ray crystal structure of the C-terminal truncated human protein, Δ(185-243)apoA-I, determined to 2.2 Å resolution by Mei and Atkinson, provides the structural basis for understanding apoA-I destabilization in amyloidosis. The sites of amyloidogenic mutations correspond to key positions within the largely helical four-segment bundle comprised of residues 1-120 and 144-184. Mutations in these positions disrupt the bundle structure and destabilize lipid-free apoA-I, thereby promoting its proteolysis. Moreover, many mutations place a hydrophilic or Pro group in the middle of the hydrophobic lipid-binding face of the amphipathic α-helices, which will likely shift the population distribution from HDL-bound to lipid-poor/free apoA-I that is relatively unstable and labile to proteolysis. Notably, the crystal structure shows segment L44-S55 in an extended conformation consistent with the β-strand-like geometry. Exposure of this segment upon destabilization of the four-segment bundle probably initiates the α-helix to β-sheet conversion in amyloidosis. In summary, we propose that the amyloidogenic mutations promote apoA-I proteolysis by destabilizing the protein structure not only in the lipid-free but also in the HDL-bound form, with segment L44-S55 providing a likely template for the cross-β-sheet conformation.
To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipo... more To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the R-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation ∆(209-243) does not change the R-helical content but reduces the protein stability. Truncation of a larger segment, ∆(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, ∆(198-243), leads to a significant reduction in the R-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion ∆(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, ∆(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.
In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated ... more In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated with hypertriglyceridemia. It has been shown that overexpression of human wild-type (WT) apoE4 in apoE-deficient mice induces hypertriglyceridemia. In contrast, overexpression of an apoE4 variant, apoE4-mut1 (apoE4(L261A, W264A, F265A, L268A, V269A)), does not induce hypertriglyceridemia and corrects hypercholesterolemia. Furthermore, overexpression of another variant, apoE4-mut2 (apoE4(W276A, L279A, V280A, V283A)), induces mild hypertriglyceridemia and does not correct hypercholesterolemia. To better understand how these mutations improve the function of apoE4, we investigated the conformation and stability of apoE4-mut1 and apoE4-mut2 and their binding to dimyristoyl phosphatidylcholine (DMPC) vesicles and to triglyceride (TG)-rich emulsion particles. We found that the mutations introduced in apoE4-mut1 lead to a more stable and compactly folded conformation of apoE4. These structural changes are associated with a slower rate of solubilization of DMPC vesicles by apoE4-mut1 and reduced binding of the protein to emulsion particles as compared to WT apoE4. Under conditions of apoE4 overexpression, the reduced binding of apoE4-mut1 to TG-rich lipoprotein particles may facilitate the lipolysis of these particles and may alter the conformation of the lipoprotein-bound apoE in a way that favors the efficient clearance of the lipoprotein remnants. Mutations introduced in apoE4-mut2 result in smaller structural alterations compared to those observed in apoE4-mut1. The slightly altered structural properties of apoE4-mut2 are associated with slightly reduced binding of this protein to TG-rich lipoprotein particles and milder hypertriglyceridemia as compared to WT apoE4. Human apolipoprotein E (apoE) is a key component of the lipoprotein transport system and is required for the maintenance of lipid homeostasis in the circulation and the brain (1-3). In humans, there are three natural apoE isoforms that differ from each other by amino acid substitutions at positions 112 and 158. ApoE3 (Cys-112, Arg-158) is the most common isoform; apoE2 (Cys-112, Cys-158) is associated with type III hyperlipoproteinemia, while apoE4 (Arg-112, Arg-158) is associated with high plasma cholesterol level and an increased risk for both coronary heart disease and Alzheimer's diseases (4-7). ApoE is one of the major protein constituents of triglyceride (TG)-rich chylomicrons and very low density lipoproteins
The physical properties in water of a series of 1: 1 acid-soap compounds formed from fatty acids ... more The physical properties in water of a series of 1: 1 acid-soap compounds formed from fatty acids and potassium soaps with saturated (10-18 carbons) and w-9 monounsaturated (18 carbons) hydrocarbon chains have been studied by using differential scanning calorimetry (DSC), X-ray diffraction, and direct and polarized light microscopy. DSC showed three phase transitions corresponding to the melting of crystalline water, the melting of crystalline lipid hydrocarbon chains, and the decomposition of the 1:l acid-soap compound into its parent fatty acid and soap. Low-and wide-angle X-ray diffraction patterns revealed spacings that corresponded (with increasing hydration) to acid-soap crystals, hexagonal type I1 liquid crystals, and lamellar liquid crystals. The lamellar phase swelled from bilayer repeat distances of 68 (at 45% H,O) to 303 A (at 90% HzO). Direct and polarized light micrographs demonstrated the formation of myelin figures as well as birefringent optical textures corresponding to hexagonal and lamellar mesophases. Assuming that 1: 1 potassium hydrogen dioleate and water were two components, we constructed a temperaturecomposition phase diagram. Interpretation of the data using the Gibbs phase rule showed that, a t >30% water, hydrocarbon chain melting was accompanied by decomposition of the 1:l acid-soap compound and the system changed from a two-component to a three-component system. Comparison of hydrated 1:l fatty acid/soap systems with hydrated soap systems suggests that the reduced degree of charge repulsion between polar groups causes half-ionized fatty acids in excess water to form bilayers rather than micelles. The 1:l fatty acid/soap system provides insights into the physical states formed by free fatty acids during transport and metabolism in vivo since, at p H 7.4, fatty acids in water and in phospholipid bilayers are near halfionization. ' In this paper, "fatty acids" (when enclosed by quotation marks) refers to the general class of compounds without specification of the ionization state of the carboxyl group. Thus, "fatty acids" could refer to fully un-ionized fatty acids, fully ionized fatty acids (soaps), mixtures of ionized and un-ionized species, and/or 1:l acidsoap compounds. The term fatty acids (when used without quotation marks or qualifying phrases) specifically refers to fully un-ionized (protonated) fatty acids.
Thermal and chemical unfolding of lipid-free apolipoprotein C-1 (apoC-1), a 6-kDa protein compone... more Thermal and chemical unfolding of lipid-free apolipoprotein C-1 (apoC-1), a 6-kDa protein component of very low density and high-density lipoproteins, was analyzed by far-UV CD. In neutral 1 mM Na 2 HPO 4 solutions containing 6-7 µg/mL protein, the apoC-1 monomer is ∼30% R-helical at 0-22°C and unfolds reversibly from about 22-80°C with T m) 51 (3°C and van't Hoff enthalpy ∆H v (T m)) 19 (3 kcal/mol. The apparent free energy of the monomer stabilization determined from the chemical unfolding at 0°C, ∆G(0°C)) 2.8 (0.8 kcal/mol, decreases by about 1 kcal/mol upon heating to 25°C. A small apparent heat capacity increment suggests the absence of a substantial hydrophobic core for the apoC-1 molecule. At pH 7, increasing apoC-1 concentration above 10 µg/mL leads to self-association and formation of additional R-helices that unfold upon both heating and cooling from room temperature. The CD data indicate that the high-temperature transition reflects a complete monomer unfolding and the low-temperature transition reflects oligomer dissociation into stable monomers. This suggests the importance of hydrophobic interactions for apoC-1 self-association. Close proximity between the highand low-temperature transitions and the absence of a plateau in the chemical unfolding curves recorded from oligomeric apoC-1 indicate marginal oligomer stability and suggest that in vivo apoC-1 transfer is mediated via the complexes with other apolipoproteins and/or lipids.
Hypertriglyceridemia (HTG) is a common lipid abnormality in humans. However, its etiology remains... more Hypertriglyceridemia (HTG) is a common lipid abnormality in humans. However, its etiology remains largely unknown. It was shown that severe HTG can be induced in mice by overexpression of wild-type (WT) apolipoprotein E (apoE) or specific apoA-I mutants. Certain mutations in apoE4 were found to affect plasma triglyceride (TG) levels in mice overexpressing the protein. HTG appeared to positively correlate with the ability of the apoE4 variants to bind to TG-rich particles, protein destabilization, and the exposure of protein hydrophobic surface in solution. Here, we propose that the apoA-I mutations that cause HTG may also lead to changes in the conformation and stability that promote binding of apoA-I to TG-rich lipoproteins. To test this hypothesis, we studied binding to TG-rich emulsion and biophysical properties of the apoA-I mutants that induce HTG, apoA-I[E110A/E111A] and apoA-I[Δ(61-78)], and compared them to those of WT apoA-I and another apoA-I mutant, apoA-I[Δ(89-99)], that does not induce HTG but causes hypercholesterolemia in mice. We found that the apoAI[E110A/E111A] and apoA-I[Δ(61-78)] mutations lead to enhanced binding of apoA-I to TG-rich particles, destabilization, and greater exposure of the hydrophobic surface of the protein. The apoA-I[Δ(89-99)] mutant did not show enhanced binding to the emulsion or a more exposed hydrophobic surface. Thus, like apoE4, the apoA-I variants that cause HTG in mice have the altered conformation and stability that facilitate their binding to TG-rich lipoproteins and thereby may lead to the reduced level of lipolysis of these lipoproteins. While many factors may be involved in induction of HTG, we suggest that an increased level of association of destabilized loosely folded apolipoproteins with TG-rich lipoproteins may contribute to some cases of HTG in humans.
Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins th... more Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into ~ 60% α-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46 residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10, 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far ultraviolet circular dichroism spectra show that [198-243] apoA-I is also unfolded in aqueous solution. However, self-association induces ~ 50% α-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid mimicking detergents, above their CMC, ~ 60% α-helix was induced in the peptide. In contrast, SDS, an anionic lipid mimicking detergent, induced helical folding in the peptide at a concentration of ~ 0.003% (1 00 μM), ~ 70 fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solublize dimyristoyl phosphatidyl choline (DMPC) liposomes and fold into ~ 60% αhelix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy, demonstrated that the peptide binds to DMPC with high affinity to form at least two sizes of relatively homogenous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide w/w, diameter and density) of both complexes are similar to those of plasma A-I/DMPC formed under similar conditions: small discoidal complexes (~ 3:1 w/w, ~ 110Å and ~ 1.10g/cm 3) formed at initial 1:1 w/w ratio and larger discoidal complexes (~ 4.6:1 w/w, ~ 165 Å and ~ 1.085g/cm 3) formed at initial 4:1 w/w ratio. The cross-linking of the peptide on the two sizes of disks is consistent with the calculated peptide numbers per particle, which result in sufficient helix to surround the lipid bilayer twice. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this Cterminal peptide model can mimic the interaction with phospholipid of plasma apoA-I to form two sizes of homogenous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma. The plasma concentration of high-density lipoprotein (HDL) is an inverse marker of potential cardiovascular disease. The well-documented anti-atherogenic function of HDL is related to its critical role in reverse cholesterol transport, such as mediation of cholesterol efflux from macrophage cells and inhibition of foam cell formation. Apolipoprotein A-I (apoA-I), an
To probe the structure and stability of the central region of lipid-free apolipoprotein (apo) A-I... more To probe the structure and stability of the central region of lipid-free apolipoprotein (apo) A-I (residues 123-165), we studied the effects of four mutations made in this region on the conformation, stability, dimyristoylphosphatidylcholine (DMPC) binding kinetics, and size of discoidal reconstituted high-density lipoprotein (rHDL) particles. The apoA-I deletion ∆(144-165) leads to a red shift in the wavelength of maximum fluorescence and a reduction in the R-helical content, the stability, the initial rate of association with DMPC liposomes, and the size of the discoidal particles. The data are consistent with the helical structure of residues 144-165, and the deletion appears to perturb the tertiary organization of the N-terminal half of apoA-I. In contrast, the deletion of the adjacent region, ∆(136-143), leads to stabilization without altering the number of residues in the helical conformation or the initial rate of association with DMPC liposomes. The quadruple substitution E125K/E128K/K133E/E139K leads to ∼17 additional residues in the helical conformation and an increase in the stability, the initial rate of association with DMPC liposomes, and the size of the rHDL particles. The findings are consistent with the disordered structure of the segment of residues 123-142, which becomes helical as a result of the quadruple mutation or upon lipid binding. The naturally occurring mutation L141R (also associated with coronary heart disease) that is located in this segment does not change the protein conformation but leads to a reduced stability and a decreased rate of association with DMPC liposomes that may relate to the observed altered functions of this mutant.
'Triacylglycerols, which usually contain at least one unsaturated fatty acid, are the most import... more 'Triacylglycerols, which usually contain at least one unsaturated fatty acid, are the most important forms of stored biological lipids in teleosts, mammals, and most plants. Since the physical properties of such mixed-chain triacylglycerols are poorly understood, a systematic study of such compounds has been initiated. Stereospecific 1,2-dioleoyl-3-acyl-sn-glycerols were synthesized with even carbon saturated fatty acyl chains of 14-24 carbons in length. Their polymorphic behavior was examined by differential scanning calorimetry and X-ray powder diffraction. The thermal behavior revealed from one to four major polymorphic transitions depending upon saturated chain length. Plots of enthalpy of fusion and entropy vs. carbon number for qelting of the most stable polymorph were linear throughout the series with slopes of 1 .O kcal/mol per carbon atom and 2.6 cal/(mol K) per carbbn atom, respectively. These slopes indicate that the saturated chains are packed in a well-ordered tightly packed lattice. When the compounds were rapidly cooled to 5 O C , X-ray powder diffraction revealed strong p' (ca. 3.8 and 4.2 A) reflections and weak /3 (ca. 4.6 A) reflections. The /3 subcell reflections intensified when the compounds were heated to within 5 O C of the meltihg temperature of the highest melting polymorph. Evidence of an 01 phase was not seen on 30-min X-ray exposures for any of the compounds. In the proposed packing arrangement the saturated and unsaturated chains are segregated into layers. The stable form of all compounds exhibits a triple layer packing mode in which a bilayer of oleoyl chains is segregated from an interdigitated layer of saturated chains. The thickness of the glycerol backbone normal to the base plane is about 4.1 A. The saturated chains are tilted 55.8' with.respect to the base plane. This tilt is close to the value of 56.5O obtained from the single crystal study of oleic acid, which may indicate that the saturated chains project from the glycerol backbone like a linear extension of the first nine carbons of the oleoyl chain. In addition, a metastable six-layer packing mode was identified for 1,2-dioleoyl-3-myristoyl-sn-glycerol. While some of the complex polymorphisms involve adjustments in chain packing, others may relate to transitions from unstable six-layer to stable three-layer structures.
Apolipoprotein A-I is the major protein in high-density lipoprotein (HDL) and plays an important ... more Apolipoprotein A-I is the major protein in high-density lipoprotein (HDL) and plays an important role during the process of reverse cholesterol transport (RCT). Knowledge of the high-resolution structure of full-length apoA-I is vital for a molecular understanding of the function of HDL at the various steps of the RCT pathway. Due to the flexible nature of apoA-I and aggregation properties, the structure of full-length lipid-free apoA-I has evaded description for over three decades. Sequence analysis of apoA-I suggested that the amphipathic α-helix is the structural motif of exchangeable apolipoprotein, and NMR, X-ray and MD simulation studies have confirmed this. Different laboratories have used different methods to probe the secondary structure distribution and organization of both the lipid-free and lipid-bound apoA-I structure. Mutation analysis, synthetic peptide models, surface chemistry and crystal structures have converged on the lipid-free apoA-I domain structure and functi...
We found earlier that apoA-I variants that induced hypertriglyceridemia (HTG) in mice had increas... more We found earlier that apoA-I variants that induced hypertriglyceridemia (HTG) in mice had increased affinity to TG-rich lipoproteins and thereby impaired their catabolism. Here, we tested whether a naturally occurring human apoA-I mutation, Lys107del, associated with HTG also promotes apoA-I binding to TG-rich particles. We expressed apoA-I[Lys107del] variant in Escherichia coli, studied its binding to TG-rich emulsion particles, and performed a physicochemical characterization of the protein. Compared with WT apoA-I, apoA-I[Lys107del] showed enhanced binding to TG-rich particles, lower stability, and greater exposure of hydrophobic surfaces. The crystal structure of truncated, Δ(185-243), apoA-I suggests that deletion of Lys107 disrupts helix registration and disturbs a stabilizing salt bridge network in the N-terminal helical bundle. To elucidate the structural changes responsible for the altered function of apoA-I[Lys107del], we studied another mutant, apoA-I [Lys107Ala]. Our fin...
CONTENTS I. INTRODUCTION II. CHEMICAL FORMULATION AND NOMENCLATURE III. PHYSICAL STATES OF CHOLES... more CONTENTS I. INTRODUCTION II. CHEMICAL FORMULATION AND NOMENCLATURE III. PHYSICAL STATES OF CHOLESTERYL ESTERS A. Crystalline states 1. Monolayer type I 2. Monolayer type II 3. Bilayer B. The mesomorphic state C. The liquid state IV. TRANSITION BETWEEN STATES V. PHASE BEHAVIOR OF CHOLESTERYL ESTERS FROM POLARIZING MICROSCOPY AND DIFFERENTIAL SCANNING CALORIMETRY A. Effects of chain length in saturated and unsaturated cholesteryl esters B. Influence of the unsaturation in the fatty acyl chain 1. Effect of double bond position 2. Effect of increasing unsaturation VISCOSITY, FLUIDITY AND MOLECULAR MOTIONS CRYSTAL AND SMECTIC MESOPHASE STRUCTURE OF CHOLESTERYL
Proceedings of the National Academy of Sciences, 1996
Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in th... more Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximately 7, by using differential scanning calorimetry and circular dichroism. At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint Tm 57 +/- 1 degree C, calorimetric enthalpy deltaH(Tm)= 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy deltaHv(Tm) approximately 32.5 kcal/mol, and cooperativity deltaHv(Tm)/deltaH(Tm) approximately 0.16. The enthalpy deltaH(Tm) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximately 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal non...
properties of apoA-I arise primarily through its important roles in the pathway of reverse choles... more properties of apoA-I arise primarily through its important roles in the pathway of reverse cholesterol transport; where apoA-I stabilizes and maintains the structure of HDL particles, promotes cellular cholesterol effl ux by binding to specifi c ATP binding cassette transporters, interacts with the enzyme lecithin:cholesterol acyltransferase (LCAT) to drive the maturation of HDL particles, binds and modifi es the lipoprotein surface to facilitate enzyme reactions, and interacts with the scavenger receptor class B type 1 for selective cholesterol ester (CE) uptake by the liver for excretion (2-4). ApoA-I exists in lipid-free, lipid-poor, and lipid-bound states in plasma, and exchanges among HDL and TAGrich lipoprotein particles like very low density lipoproteins (VLDLs) and chylomicrons (CMs) (5). ApoA-I interacts primarily with the hydrocarbon chains of the phospholipids (PL) on discoidal and spherical HDLs (6-8) and may also interact with the hydrophobic CE core of spherical HDL and the hydrophobic TAG core of TAG-rich lipoproteins. The conformational fl exibility of apoA-I allows it to adopt a variety of conformations involving lipid adsorption, partial or full desorption, and fl exible unfolding and refolding in diverse physical environments to facilitate its multiple functions. Studies of the molecular mechanisms of the lipid association and the conformational fl exibility of apoA-I are essential for understanding the structure-function relationships. ApoA-I has an exon 3 encoded region (residues 1-43), and an exon 4 encoded region (residues 44-243) that contains 10 11/22-mer tandem repeat amino acid segments that are predicted to form class A and class Y amphipathic ␣-helices (A ␣ Hs) (9, 10). These A ␣ Hs (helix 1-10) are the lipid binding motif of apoA-I and the structural basis for its multiple functions. Segment deletion and point mutation studies have elucidated the possible conformation and functions for each helical segment (11-16). Several studies have suggested that the lipid-free apoA-I molecule possesses a Abstract Apolipoprotein A-I (apoA-I) has a great conformational fl exibility to exist in lipid-free, lipid-poor, and lipid-bound states during lipid metabolism. To address the lipid binding and the dynamic desorption behavior of apoA-I at lipoprotein surfaces, apoA-I, ⌬ (185-243)apoA-I, and ⌬ (1-59)(185-243)apoA-I were studied at triolein/water and phosphatidylcholine/triolein/water interfaces with special attention to surface pressure. All three proteins are surface active to both interfaces lowering the interfacial tension and thus increasing the surface pressure to modify the interfaces. ⌬ (185-243)apoA-I adsorbs much more slowly and lowers the interfacial tension less than full-length apoA-I, confi rming that the C-terminal domain (residues 185-243) initiates the lipid binding. ⌬ (1-59)(185-243)apoA-I binds more rapidly and lowers the interfacial tension more than ⌬ (185-243)apoA-I, suggesting that destabilizing the N-terminal ␣-helical bundle (residues 1-185) restores lipid binding. The three proteins desorb from both interfaces at different surface pressures revealing that different domains of apoA-I possess different lipid affi nity. ⌬ (1-59)(185-243)apoA-I desorbs at lower pressures compared with apoA-I and ⌬ (185-243) apoA-I indicating that it is missing a strong lipid association motif. We propose that during lipoprotein remodeling, surface pressure mediates the adsorption and partial or full desorption of apoA-I allowing it to exchange among different lipoproteins and adopt various conformations to facilitate its multiple functions.
that raise HDL levels do not necessarily confer additional cardioprotection (Refs. 1, 2 and refer... more that raise HDL levels do not necessarily confer additional cardioprotection (Refs. 1, 2 and references therein). Plasma HDL carries from two to fi ve copies of apoA-I depending on the particle size (8-12 nm) and varies in particle shape (nascent "discoidal" or mature "spherical", Fig. 1), protein conformation, biochemical composition, and functional properties (1). The nascent HDL particle is generally envisioned as a contorted disc composed of a cholesterolcontaining phospholipid bilayer that is encircled by two antiparallel apoA-I molecules in a highly ␣-helical dynamic "double-belt" conformation (3-5). Micelle-like lipid packing has also been proposed in some studies (Ref. 6 and references therein). ApoA-I on HDL activates LCAT that converts nascent HDL into mature spherical particles containing a core of apolar lipids, mainly cholesterol esters, and a small amount of triacylglycerides. ApoA-I on mature HDL is proposed to form a double belt similar to that on nascent HDL but bent in a "trefoil/tetrafoil" fashion to confer 2D curvature to the surface of spherical particles (7, 8) (Fig. 1B). ApoA-I on the HDL surface forms a fl exible structural scaffold and is an important ligand for many functional interactions that direct HDL metabolism (1, 7-11). The amino acid sequence of apoA-I starts with a G-repeat (residues 1-43) followed by ten Pro-punctuated tandem Abstract HDL removes cell cholesterol and protects against atherosclerosis. ApoA-I provides a fl exible structural scaffold and an important functional ligand on the HDL surface. We propose structural models for apoA-I Milano (R173C) and apoA-I Paris (R151C) mutants that show high cardioprotection despite low HDL levels. Previous studies established that two apoA-I molecules encircle HDL in an antiparallel, helical double-belt conformation. Recently, we solved the atomic structure of lipid-free ⌬ (185-243)apoA-I and proposed a conformational ensemble for apoA-I WT on HDL. Here we modify this ensemble to understand how intermolecular disulfi des involving C173 or C151 infl uence protein conformation. The double-belt conformations are modifi ed by belt rotation, main-chain unhinging around Gly, and Pro-induced helical bending, and they are verifi ed by comparison with previous experimental studies and by molecular dynamics simulations of apoA-I Milano homodimer. In our models, the molecular termini repack on various-sized HDL, while packing around helix-5 in apoA-I WT , helix-6 in apoA-I Paris , or helix-7 in apoA-I Milano homodimer is largely conserved. We propose how the disulfi de-induced constraints alter the protein conformation and facilitate dissociation of the C-terminal segment from HDL to recruit additional lipid. Our models unify previous studies of apoA-I Milano and demonstrate how the mutational effects propagate to the molecular termini, altering their conformations, dynamics, and function.-Gursky, O.
The primary and secondary structure of human plasma apolipoprotein A-I and apolipoprotein E-3 hav... more The primary and secondary structure of human plasma apolipoprotein A-I and apolipoprotein E-3 have been analyzed to further our understanding of the secondary and tertiary conformation of these proteins and the structure and function of plasma lipoprotein particles. The methods used to analyze the primary sequence of these proteins used computer programs: (a) to identify repeated patterns within these proteins on the basis of conservative substitutions and similarities within the physicochemical properties of each residue; (b) for local averaging, hydrophobic moment, and Fourier analysis of the physicochemical properties; and (c) for secondary structure prediction of each protein carried out using homology, statistical, and information theory based methods. Circular dichroism was used to study purified lipid-protein complexes of each protein and quantitate the secondary structure in a lipid environment. The data from these analyses were integrated into a single secondary structure prediction to derive a model of each protein. The sequence homology within apolipoproteins A-I, E-3, and A-IV is used to derive a consensus sequence for two 11 amino acid repeating sequences in this family of proteins.
Apolipoprotein A-I (apoA-I) is the main protein of plasma high-density lipoproteins (HDL, or good... more Apolipoprotein A-I (apoA-I) is the main protein of plasma high-density lipoproteins (HDL, or good cholesterol) that remove excess cell cholesterol and protect against atherosclerosis. In hereditary amyloidosis, mutations in apoA-I promote its proteolysis and the deposition of the 9-11 kDa N-terminal fragments as fibrils in vital organs such as kidney, liver, and heart, causing organ damage. All known amyloidogenic mutations in human apoA-I are clustered in two residue segments, 26-107 and 154-178. The X-ray crystal structure of the C-terminal truncated human protein, Δ(185-243)apoA-I, determined to 2.2 Å resolution by Mei and Atkinson, provides the structural basis for understanding apoA-I destabilization in amyloidosis. The sites of amyloidogenic mutations correspond to key positions within the largely helical four-segment bundle comprised of residues 1-120 and 144-184. Mutations in these positions disrupt the bundle structure and destabilize lipid-free apoA-I, thereby promoting its proteolysis. Moreover, many mutations place a hydrophilic or Pro group in the middle of the hydrophobic lipid-binding face of the amphipathic α-helices, which will likely shift the population distribution from HDL-bound to lipid-poor/free apoA-I that is relatively unstable and labile to proteolysis. Notably, the crystal structure shows segment L44-S55 in an extended conformation consistent with the β-strand-like geometry. Exposure of this segment upon destabilization of the four-segment bundle probably initiates the α-helix to β-sheet conversion in amyloidosis. In summary, we propose that the amyloidogenic mutations promote apoA-I proteolysis by destabilizing the protein structure not only in the lipid-free but also in the HDL-bound form, with segment L44-S55 providing a likely template for the cross-β-sheet conformation.
To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipo... more To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the R-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation ∆(209-243) does not change the R-helical content but reduces the protein stability. Truncation of a larger segment, ∆(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, ∆(198-243), leads to a significant reduction in the R-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion ∆(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, ∆(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.
In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated ... more In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated with hypertriglyceridemia. It has been shown that overexpression of human wild-type (WT) apoE4 in apoE-deficient mice induces hypertriglyceridemia. In contrast, overexpression of an apoE4 variant, apoE4-mut1 (apoE4(L261A, W264A, F265A, L268A, V269A)), does not induce hypertriglyceridemia and corrects hypercholesterolemia. Furthermore, overexpression of another variant, apoE4-mut2 (apoE4(W276A, L279A, V280A, V283A)), induces mild hypertriglyceridemia and does not correct hypercholesterolemia. To better understand how these mutations improve the function of apoE4, we investigated the conformation and stability of apoE4-mut1 and apoE4-mut2 and their binding to dimyristoyl phosphatidylcholine (DMPC) vesicles and to triglyceride (TG)-rich emulsion particles. We found that the mutations introduced in apoE4-mut1 lead to a more stable and compactly folded conformation of apoE4. These structural changes are associated with a slower rate of solubilization of DMPC vesicles by apoE4-mut1 and reduced binding of the protein to emulsion particles as compared to WT apoE4. Under conditions of apoE4 overexpression, the reduced binding of apoE4-mut1 to TG-rich lipoprotein particles may facilitate the lipolysis of these particles and may alter the conformation of the lipoprotein-bound apoE in a way that favors the efficient clearance of the lipoprotein remnants. Mutations introduced in apoE4-mut2 result in smaller structural alterations compared to those observed in apoE4-mut1. The slightly altered structural properties of apoE4-mut2 are associated with slightly reduced binding of this protein to TG-rich lipoprotein particles and milder hypertriglyceridemia as compared to WT apoE4. Human apolipoprotein E (apoE) is a key component of the lipoprotein transport system and is required for the maintenance of lipid homeostasis in the circulation and the brain (1-3). In humans, there are three natural apoE isoforms that differ from each other by amino acid substitutions at positions 112 and 158. ApoE3 (Cys-112, Arg-158) is the most common isoform; apoE2 (Cys-112, Cys-158) is associated with type III hyperlipoproteinemia, while apoE4 (Arg-112, Arg-158) is associated with high plasma cholesterol level and an increased risk for both coronary heart disease and Alzheimer's diseases (4-7). ApoE is one of the major protein constituents of triglyceride (TG)-rich chylomicrons and very low density lipoproteins
The physical properties in water of a series of 1: 1 acid-soap compounds formed from fatty acids ... more The physical properties in water of a series of 1: 1 acid-soap compounds formed from fatty acids and potassium soaps with saturated (10-18 carbons) and w-9 monounsaturated (18 carbons) hydrocarbon chains have been studied by using differential scanning calorimetry (DSC), X-ray diffraction, and direct and polarized light microscopy. DSC showed three phase transitions corresponding to the melting of crystalline water, the melting of crystalline lipid hydrocarbon chains, and the decomposition of the 1:l acid-soap compound into its parent fatty acid and soap. Low-and wide-angle X-ray diffraction patterns revealed spacings that corresponded (with increasing hydration) to acid-soap crystals, hexagonal type I1 liquid crystals, and lamellar liquid crystals. The lamellar phase swelled from bilayer repeat distances of 68 (at 45% H,O) to 303 A (at 90% HzO). Direct and polarized light micrographs demonstrated the formation of myelin figures as well as birefringent optical textures corresponding to hexagonal and lamellar mesophases. Assuming that 1: 1 potassium hydrogen dioleate and water were two components, we constructed a temperaturecomposition phase diagram. Interpretation of the data using the Gibbs phase rule showed that, a t >30% water, hydrocarbon chain melting was accompanied by decomposition of the 1:l acid-soap compound and the system changed from a two-component to a three-component system. Comparison of hydrated 1:l fatty acid/soap systems with hydrated soap systems suggests that the reduced degree of charge repulsion between polar groups causes half-ionized fatty acids in excess water to form bilayers rather than micelles. The 1:l fatty acid/soap system provides insights into the physical states formed by free fatty acids during transport and metabolism in vivo since, at p H 7.4, fatty acids in water and in phospholipid bilayers are near halfionization. ' In this paper, "fatty acids" (when enclosed by quotation marks) refers to the general class of compounds without specification of the ionization state of the carboxyl group. Thus, "fatty acids" could refer to fully un-ionized fatty acids, fully ionized fatty acids (soaps), mixtures of ionized and un-ionized species, and/or 1:l acidsoap compounds. The term fatty acids (when used without quotation marks or qualifying phrases) specifically refers to fully un-ionized (protonated) fatty acids.
Thermal and chemical unfolding of lipid-free apolipoprotein C-1 (apoC-1), a 6-kDa protein compone... more Thermal and chemical unfolding of lipid-free apolipoprotein C-1 (apoC-1), a 6-kDa protein component of very low density and high-density lipoproteins, was analyzed by far-UV CD. In neutral 1 mM Na 2 HPO 4 solutions containing 6-7 µg/mL protein, the apoC-1 monomer is ∼30% R-helical at 0-22°C and unfolds reversibly from about 22-80°C with T m) 51 (3°C and van't Hoff enthalpy ∆H v (T m)) 19 (3 kcal/mol. The apparent free energy of the monomer stabilization determined from the chemical unfolding at 0°C, ∆G(0°C)) 2.8 (0.8 kcal/mol, decreases by about 1 kcal/mol upon heating to 25°C. A small apparent heat capacity increment suggests the absence of a substantial hydrophobic core for the apoC-1 molecule. At pH 7, increasing apoC-1 concentration above 10 µg/mL leads to self-association and formation of additional R-helices that unfold upon both heating and cooling from room temperature. The CD data indicate that the high-temperature transition reflects a complete monomer unfolding and the low-temperature transition reflects oligomer dissociation into stable monomers. This suggests the importance of hydrophobic interactions for apoC-1 self-association. Close proximity between the highand low-temperature transitions and the absence of a plateau in the chemical unfolding curves recorded from oligomeric apoC-1 indicate marginal oligomer stability and suggest that in vivo apoC-1 transfer is mediated via the complexes with other apolipoproteins and/or lipids.
Hypertriglyceridemia (HTG) is a common lipid abnormality in humans. However, its etiology remains... more Hypertriglyceridemia (HTG) is a common lipid abnormality in humans. However, its etiology remains largely unknown. It was shown that severe HTG can be induced in mice by overexpression of wild-type (WT) apolipoprotein E (apoE) or specific apoA-I mutants. Certain mutations in apoE4 were found to affect plasma triglyceride (TG) levels in mice overexpressing the protein. HTG appeared to positively correlate with the ability of the apoE4 variants to bind to TG-rich particles, protein destabilization, and the exposure of protein hydrophobic surface in solution. Here, we propose that the apoA-I mutations that cause HTG may also lead to changes in the conformation and stability that promote binding of apoA-I to TG-rich lipoproteins. To test this hypothesis, we studied binding to TG-rich emulsion and biophysical properties of the apoA-I mutants that induce HTG, apoA-I[E110A/E111A] and apoA-I[Δ(61-78)], and compared them to those of WT apoA-I and another apoA-I mutant, apoA-I[Δ(89-99)], that does not induce HTG but causes hypercholesterolemia in mice. We found that the apoAI[E110A/E111A] and apoA-I[Δ(61-78)] mutations lead to enhanced binding of apoA-I to TG-rich particles, destabilization, and greater exposure of the hydrophobic surface of the protein. The apoA-I[Δ(89-99)] mutant did not show enhanced binding to the emulsion or a more exposed hydrophobic surface. Thus, like apoE4, the apoA-I variants that cause HTG in mice have the altered conformation and stability that facilitate their binding to TG-rich lipoproteins and thereby may lead to the reduced level of lipolysis of these lipoproteins. While many factors may be involved in induction of HTG, we suggest that an increased level of association of destabilized loosely folded apolipoproteins with TG-rich lipoproteins may contribute to some cases of HTG in humans.
Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins th... more Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into ~ 60% α-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46 residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10, 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far ultraviolet circular dichroism spectra show that [198-243] apoA-I is also unfolded in aqueous solution. However, self-association induces ~ 50% α-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid mimicking detergents, above their CMC, ~ 60% α-helix was induced in the peptide. In contrast, SDS, an anionic lipid mimicking detergent, induced helical folding in the peptide at a concentration of ~ 0.003% (1 00 μM), ~ 70 fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solublize dimyristoyl phosphatidyl choline (DMPC) liposomes and fold into ~ 60% αhelix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy, demonstrated that the peptide binds to DMPC with high affinity to form at least two sizes of relatively homogenous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide w/w, diameter and density) of both complexes are similar to those of plasma A-I/DMPC formed under similar conditions: small discoidal complexes (~ 3:1 w/w, ~ 110Å and ~ 1.10g/cm 3) formed at initial 1:1 w/w ratio and larger discoidal complexes (~ 4.6:1 w/w, ~ 165 Å and ~ 1.085g/cm 3) formed at initial 4:1 w/w ratio. The cross-linking of the peptide on the two sizes of disks is consistent with the calculated peptide numbers per particle, which result in sufficient helix to surround the lipid bilayer twice. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this Cterminal peptide model can mimic the interaction with phospholipid of plasma apoA-I to form two sizes of homogenous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma. The plasma concentration of high-density lipoprotein (HDL) is an inverse marker of potential cardiovascular disease. The well-documented anti-atherogenic function of HDL is related to its critical role in reverse cholesterol transport, such as mediation of cholesterol efflux from macrophage cells and inhibition of foam cell formation. Apolipoprotein A-I (apoA-I), an
To probe the structure and stability of the central region of lipid-free apolipoprotein (apo) A-I... more To probe the structure and stability of the central region of lipid-free apolipoprotein (apo) A-I (residues 123-165), we studied the effects of four mutations made in this region on the conformation, stability, dimyristoylphosphatidylcholine (DMPC) binding kinetics, and size of discoidal reconstituted high-density lipoprotein (rHDL) particles. The apoA-I deletion ∆(144-165) leads to a red shift in the wavelength of maximum fluorescence and a reduction in the R-helical content, the stability, the initial rate of association with DMPC liposomes, and the size of the discoidal particles. The data are consistent with the helical structure of residues 144-165, and the deletion appears to perturb the tertiary organization of the N-terminal half of apoA-I. In contrast, the deletion of the adjacent region, ∆(136-143), leads to stabilization without altering the number of residues in the helical conformation or the initial rate of association with DMPC liposomes. The quadruple substitution E125K/E128K/K133E/E139K leads to ∼17 additional residues in the helical conformation and an increase in the stability, the initial rate of association with DMPC liposomes, and the size of the rHDL particles. The findings are consistent with the disordered structure of the segment of residues 123-142, which becomes helical as a result of the quadruple mutation or upon lipid binding. The naturally occurring mutation L141R (also associated with coronary heart disease) that is located in this segment does not change the protein conformation but leads to a reduced stability and a decreased rate of association with DMPC liposomes that may relate to the observed altered functions of this mutant.
'Triacylglycerols, which usually contain at least one unsaturated fatty acid, are the most import... more 'Triacylglycerols, which usually contain at least one unsaturated fatty acid, are the most important forms of stored biological lipids in teleosts, mammals, and most plants. Since the physical properties of such mixed-chain triacylglycerols are poorly understood, a systematic study of such compounds has been initiated. Stereospecific 1,2-dioleoyl-3-acyl-sn-glycerols were synthesized with even carbon saturated fatty acyl chains of 14-24 carbons in length. Their polymorphic behavior was examined by differential scanning calorimetry and X-ray powder diffraction. The thermal behavior revealed from one to four major polymorphic transitions depending upon saturated chain length. Plots of enthalpy of fusion and entropy vs. carbon number for qelting of the most stable polymorph were linear throughout the series with slopes of 1 .O kcal/mol per carbon atom and 2.6 cal/(mol K) per carbbn atom, respectively. These slopes indicate that the saturated chains are packed in a well-ordered tightly packed lattice. When the compounds were rapidly cooled to 5 O C , X-ray powder diffraction revealed strong p' (ca. 3.8 and 4.2 A) reflections and weak /3 (ca. 4.6 A) reflections. The /3 subcell reflections intensified when the compounds were heated to within 5 O C of the meltihg temperature of the highest melting polymorph. Evidence of an 01 phase was not seen on 30-min X-ray exposures for any of the compounds. In the proposed packing arrangement the saturated and unsaturated chains are segregated into layers. The stable form of all compounds exhibits a triple layer packing mode in which a bilayer of oleoyl chains is segregated from an interdigitated layer of saturated chains. The thickness of the glycerol backbone normal to the base plane is about 4.1 A. The saturated chains are tilted 55.8' with.respect to the base plane. This tilt is close to the value of 56.5O obtained from the single crystal study of oleic acid, which may indicate that the saturated chains project from the glycerol backbone like a linear extension of the first nine carbons of the oleoyl chain. In addition, a metastable six-layer packing mode was identified for 1,2-dioleoyl-3-myristoyl-sn-glycerol. While some of the complex polymorphisms involve adjustments in chain packing, others may relate to transitions from unstable six-layer to stable three-layer structures.
Uploads
Papers by David Atkinson