Dimyristoylphosphatidylcholine Complexes Research Articles

Role of Secondary Structure in Protein−Phospholipid Surface Interactions: Reconstitution and Denaturation of Apolipoprotein C-I:DMPC Complexes

Binding of protein to a phospholipid surface is commonly mediated by amphipathic alpha-helices. To understand the role of alpha-helical structure in protein-lipid interactions, we used discoidal lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and human apolipoprotein C-I (apoC-I, 6 kDa) or its mutants containing single Pro substitutions along the sequence and differing in their alpha-helical content in solution (0-48%) and on DMPC (40-75%). Thermal denaturation revealed that lipoprotein stability correlates weakly with the protein helix content: proteins with higher alpha-helical content on DMPC may form more stable complexes. Lipoprotein reconstitution upon cooling from the heat-denatured state and DMPC clearance studies revealed that protein secondary structure in solution and on DMPC correlates strongly with the maximal temperature of lipoprotein reconstitution: more helical proteins can reconstitute lipoproteins at higher temperatures. Interestingly, at Tc = 24 degrees C of the DMPC gel-to-liquid crystal transition, the clearance rate is independent of the protein helical content. Consequently, if the packing defects at the phospholipid surface are readily available (e.g., at the lipid phase boundary), insertion of protein into these defects is independent of the secondary structure in solution. However, if hydrophobic defects are limited, protein binding and insertion are aided by other surface-bound proteins and depend on their helical propensity: the larger the propensity, the faster the binding and the broader its temperature range. This positive cooperativity in binding of alpha-helices to phospholipid surface, which may result from direct and/or lipid-mediated protein-protein interactions, may be important for lipoprotein metabolism and for protein-membrane binding.

Lipid-induced Extension of Apolipoprotein E Helix 4 Correlates with Low Density Lipoprotein Receptor Binding Ability

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

Localization of the heparin binding site in human apolipoprotein AV

Apolipoprotein (apo) A-V is a potent modulator of plasma triacylglycerol (TG) levels. The molecular basis for this activity is not understood. Recent studies have shown that apoA-V possesses heparin binding activity. A stretch of 42 amino acids (residue 186 to 227) in apoA-V has been identified by sequence analysis to contain 5 arginines, 3 lysines, 4 histidines and no negatively charged amino acids. This segment is hypothesized to be the heparin binding region of apoA-V. To test this, site directed mutagenesis was used to generate three mutations in this region, in which positively charged amino acids were replaced by either negatively charged or uncharged polar amino acids. The resulting mutatants (R210E/K211Q-apoA-V, K215Q/K217E-apoA-V, and R210E/K211Q/K215Q/K217E-apoA-V) were complexed with dimyristoylphosphatidylcholine (DMPC) and subjected to heparin Sepharose affinity chromatography and surface plasmon resonance spectroscopy using a heparin-coated sensor chip. With both methods of analysis, compared to WT apoA-V DMPC complexes, the mutants showed decreased affinity for heparin. These results show that positively charged residues between position 186 and 227 of human apoA-V contribute to heparin binding. This region of apoA-V may play an important role in cell surface interactions by binding with heparan sulfate proteogylcans, indirectly affecting lipoprotein lipase activity, possibly contributing to apoA-V’s ability to influence plasma TG levels. Supported by NIH.

Electrostatic Effects on the Stability of Discoidal High-Density Lipoproteins

High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein alpha-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na(2)SO(4) increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 degrees C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by deltaDeltaG* approximately equal 3.5 kcal/mol at 37 degrees C and increases the activation energy of their denaturation and fusion by deltaE(a) approximately equal deltaDeltaH* approximately equal 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.

Structure-guided Protein Engineering Modulates Helix Bundle Exchangeable Apolipoprotein Properties

Apolipoprotein (apo) E plays a major role in lipid metabolism by mediating cellular uptake of lipoprotein particles through interaction with members of the low density lipoprotein (LDL) receptor family. The primary region of apoE responsible for receptor binding has been limited to a cluster of basic amino acids between residues 134 and 150, located in the fourth helix of the N-terminal domain globular helix bundle structure. To investigate structural and functional requirements of this "receptor binding region" we engineered an apolipoprotein chimera wherein residues 131-151 of human apoE were substituted for residues 146-166 (helix 5) of Manduca sexta apolipophorin III (apoLp-III). Recombinant hybrid apolipoprotein was expressed in Escherichia coli, isolated, and characterized. Hybrid apolipoprotein and apoE3-N-terminal, but not apoLp-III, bound to heparin-Sepharose. Far UV circular dichroism spectroscopy revealed the presence of predominantly alpha-helix secondary structure, and stability studies revealed a urea denaturation midpoint of 1.05 m, similar to wild-type apoLp-III. Hybrid apolipoprotein-induced dimyristoylphosphatidylcholine (DMPC) bilayer vesicle solubilization activity was significantly enhanced compared with either parent protein, consistent with detection of solvent-exposed hydrophobic regions on the protein in fluorescent dye binding experiments. Unlike wild-type apoLp-III.DMPC complexes, disc particles bearing the hybrid apolipoprotein competed with 125ILDL for binding to the LDL receptor on cultured human skin fibroblasts. We conclude that a hybrid apolipoprotein containing a key receptor recognition element of apoE preserves the structural integrity of the parent protein while conferring a new biological activity, illustrating the potential of helix swapping to introduce desirable biological properties into unrelated or engineered apolipoproteins.

An N-terminal three-helix fragment of the exchangeable insect apolipoprotein apolipophorin III conserves the lipid binding properties of wild-type protein.

Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.

Membrane-bound states of alpha-lactalbumin: implications for the protein stability and conformation.

alpha-Lactalbumin (alpha-LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC alpha-LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC-alpha-LA was sensitive to two thermal transitions: the Tc of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein-DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). On the other hand, above the protein transition (70 degrees C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC-alpha-LA complex showed a distinct fluorescence thermal transition between 40 and 60 degrees C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan-containing surface is covered by the lipid phase of the bilayer.

Comparison of lipid-binding and lecithin:cholesterol acyltransferase activation of the amino- and carboxyl-terminal domains of human apolipoprotein E3.

To extend the characterization of the functional domains of apolipoprotein E (apoE), the amino-(residues 1-191, 22-kDa) and carboxyl-terminal (residues 216-299, 10-kDa) fragments were tested for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. A disulfide bond linking helices 2 and 3 of the four-helix bundle amino-terminal domain was introduced by mutating threonine-57 to cysteine (Thr57-->Cys) in apoE3 (cysteine at position 112) to determine the influence of the disulfide bond on the properties of this domain. Lipid-binding properties were determined by the ability to form complexes with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine, assessed by measuring decreases in turbidity as a function of temperature. The results demonstrate that the relative lipid binding efficiencies were intact apoE3 approximately 10-kDa fragment > 22-kDa fragment > Thr57-->Cys variant. In addition, free, non-lipid-associated protein was observed with the two 22-kDa fragments but not with intact apoE3 or the 10-kDa fragment. The transition temperatures determined by fluorescence polarization were higher for the DMPC complexes with intact apoE3 and with 22- and 10-kDa fragments (25.5 degrees C) than with the 22-kDa Thr57-->Cys variant (23.5 degrees C), suggesting that the variant fragment possessed the lowest affinity for lipid. Attenuated total reflection infrared measurements of the complexes indicated that the long axes of the alpha-helices of the various apoE forms were parallel to the acyl chains of the phospholipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors affecting the stability and conformation of Locusta migratoria apolipophorin III.

Apolipophorin III (apoLp-III) from the migratory locust, Locusta migratoria, represents the only full-length apolipoprotein whose three-dimensional structure has been solved. In the present study, spectroscopic methods have been employed to investigate the effects of deglycosylation (via endoglycosidase F treatment) and complexation with lipid on the stability and conformation of this protein. Addition of isolated lipid-free apoLp-III to sonicated vesicles of dimyristoylphosphatidylcholine (DMPC) resulted in the formation of relatively uniform disklike complexes with an average Strokes diameter of 13.5 nm. Flotation equilibrium experiments conducted in the analytical ultracentrifuge revealed a particle molecular mass of 588 500 Da. Chemical cross-linking and compositional analysis of apoLp-III.DMPC complexes indicated five apoLp-III molecules per disk and an overall DMPC:apoLp-III molar ratio of 122:1. Circular dichroism (CD) spectra of apoLp-III samples suggested a loss of alpha-helical structure upon deglycosylation, while complexation with DMPC did not significantly alter the helix content (estimated to be > 75%). Fluorescence spectroscopy revealed that the apoLp-III tryptophan fluorescence emission maximum was blue-shifted from 347 to 332 and 321 nm upon deglycosylation and complexation with DMPC, respectively. In quenching experiments with native apoLp-III, tryptophan residues were shielded from the positively charged quencher, CsCl. Increased exposure to KI, CsCl, and acrylamide was observed upon deglycosylation, whereas complexation with DMPC yielded lower Ksv values for KI and acrylamide and an increased value for CsCl versus native lipid-free apoLp-III. In guanidine hydrochloride denaturation studies monitored by CD or fluorescence, native, lipid-free apoLp-III displayed a denaturation midpoint of 0.60 M, and delta GDH2O = 5.37 kcal/mol was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of insect apolipophorin III to dimyristoylphosphatidylcholine vesicles. Evidence for a conformational change.

Apolipophorin-III (apoLp-III), a hemolymph protein of Manduca sexta, can reversibly associate with the surface of lipoprotein particles. In order to examine the lipid-associated form of apoLp-III, the present studies investigate the structure and properties of apoLp-III complexes with dimyristoylphosphatidylcholine (DMPC). Association of apoLp-III with DMPC vesicles results in the formation of uniform discs with an average diameter and width of 18.5 +/- 2.0 nm and 4.8 +/- 0.8 nm, respectively, as determined by electron microscopy. ApoLp-III.DMPC complexes analyzed by pore-limiting native gradient PAGE demonstrated that a single major species of complex was formed within a wide range of lipid to protein molar ratios (DMPC:apoLp-III; 13:1 to 360:1). Flotation equilibrium experiments, conducted in an analytical ultracentrifuge, confirmed that only one species of apoLp-III.DMPC complex was formed at an initial lipid to protein molar ratio of 67:1, with an apparent molecular mass of 642,000. Complexes cross-linked with dimethyl suberimidate indicate that there are a maximum of 6 apoLp-III molecules per disc. Circular dichroism experiments revealed that apoLp-III becomes essentially completely alpha-helical on formation of apoLp-III.DMPC complexes. Compared to apoLp-III in the lipid-free state, apoLp-III.DMPC complexes were relatively resistant to denaturation by guanidine HCl, displaying denaturation transitions with midpoints at 2.2 and 3.7 M guanidine HCl, respectively. The fluorescence excitation and emission spectra of apoLp-III.DMPC complexes demonstrate a large enhancement of tyrosine fluorescence as compared to the lipid-free state, suggesting that a conformational change occurs when apoLp-III associates with a lipid surface. Denaturation of apoLp-III in the complex by guanidine HCl resulted in a tyrosine fluorescence level similar to that of lipid-free apoLp-III in the presence of guanidine HCl. The tyrosine-induced fluorescence of the complex was quenched with both Cs+ (Kq = 0.573 M-1) and KI (Kq = 0.376 M-1). The results presented in this study indicate that the conformation of apoLp-III is stabilized when complexed with phospholipids and suggest that tyrosine fluorescence provides a sensitive method to detect M. sexta apoLp-III interaction with lipid surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylated low density lipoprotein reduces its ligand activity for the scavenger receptor after interaction with reconstituted high density lipoprotein.

Complexes of apolipoprotein A-I (apoA-I) with phospholipids are known to induce cholesterol efflux from cells. In a cholesteryl ester accumulation system in which rat peritoneal macrophages were incubated with acetylated low density lipoprotein (acetyl-LDL) and either dimyristoylphosphatidylcholine complexes (DMPC/apoA-I) or native high density lipoprotein (HDL), DMPC/apoA-I exhibited a much stronger effect than native HDL in preventing cholesteryl ester accumulation. The mechanism for this phenomenon was investigated in the present study. After 18 h incubation with DMPC/apoA-I in a cell-free system, acetyl-LDL was re-isolated from DMPC/apoA-I by Sephacryl S-300 gel filtration chromatography. Re-isolated acetyl-LDL exhibited an increase in its phospholipid content by 86% as well as a reduction in the electrophoretic mobility. Its endocytic degradation by macrophages was reduced by 60% when compared with control acetyl-LDL, suggesting a significant reduction in the ligand activity for the macrophage scavenger receptor. Transfer of apolipoproteins between acetyl-LDL and DMPC/apoA-I did not occur. These results indicate that transfer of DMPC from DMPC/apoA-I to acetyl-LDL weakens the ligand activity for the scavenger receptor due probably to a decrease in net negative charge. This study demonstrated for the first time that lipid modification (change in the lipid moiety) of acetyl-LDL can induce alteration in its apolipoprotein moiety, leading to a significant loss of its biological activity. Because discoidal HDLs are known to occur in vivo, this phenomenon may explain one of the anti-atherogenic functions of HDL in vivo.

Interaction of reconstituted high density lipoprotein discs containing human apolipoprotein A-I (ApoA-I) variants with murine adipocytes and macrophages. Evidence for reduced cholesterol efflux promotion by apoA-I(Pro165–>Arg).

Interaction of cells with both native and reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I mediates efflux of cellular cholesterol. To characterize structural requirements for this activity in apoA-I, six different genetic apoA-I variants and the corresponding normal allele products were isolated from heterozygous carriers, reconstituted with dimyristoylphosphatidylcholine (DMPC) into discoidal HDL and compared with regard to their ability to release intracellular cholesterol from murine adipocytes and peritoneal macrophages. In previous studies we determined the amino acid changes of these variants: Pro3-->Arg, Pro4-->Arg, Lys107-->0, Lys107-->Met, Pro165-->Arg, and Glu198-->Lys (von Eckardstein, A., Funke, H., Walter, M., Altland, K., Benninghoven, A., and Assmann, G. (1990) J. Biol. Chem. 265, 8610-8617) and demonstrated that all apoA-I variants except apoA-I(Lys107-->0) form discoidal HDL particles with phosphatidylcholine analogues indistinguishable from normal apoA-I (Jonas, A., von Eckardstein, A., Kezdy, K. E., Steinmetz, A., and Assmann, G. (1991) J. Lipid Res. 32, 95-106). In the present study, all apoA-I.DMPC complexes except those containing apoA-I(Pro165-->Arg) promoted cholesterol efflux as effectively as those containing normal apoA-I. Cholesterol efflux from adipocytes obtained after 180 min of incubation with apoA-I(Pro165-->Arg).DMPC complexes was decreased by 30% although this variant was bound to adipocytes with normal affinity. By contrast, apoA-I(Lys107-->Met).DMPC complexes were decreased in their binding affinity compared to normal apoA-I.DMPC complexes but normally promoted cholesterol efflux. Incubation of mouse peritoneal macrophages with apoA-I(Pro165-->Arg).DMPC complexes did also result in a 30% decreased efflux of radiolabeled cholesterol if compared to rHDL with the normal allele product from the same donor. Together the observations suggest that the substitution of proline at residue 165 interferes with the formation of a structural domain in apoA-I that is crucial for cellular cholesterol efflux stimulation. Furthermore, our results suggest that binding of HDL to adipocytes and cholesterol efflux promotion by HDL requires different structural domains.

Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.

There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.

Reversible disc-micellization of dimyristoylphosphatidylcholine bilayers induced by melittin and [Ala-14]melittin

The properties of melittin and a synthetic analogue, [Ala-14]melittin (P14A), in inducing reversible transitions between vesicles and micelles at the liquid-crystalline to gel phase transition temperature ( T m) in complexes with saturated phosphatidylcholines has been studied by deuterium NMR and freeze-fracture electron microscopy (EM). At concentrations between 3 and 5 mol% relative to lipid, each peptide causes reversible micellization of dimyristoylphosphatidylcholine (DMPC) bilayers when the temperature is lowered below T m. At concentrations of 5 mol% relative to lipid, the peptides induce macroscopic magnetic orientation of DMPC bilayers at temperatures around the centre of the lipid phase transition; at temperatures a few degrees above T m, magnetic orientation is lost. These effects suggest a progressive phase separation of peptide and lipid on cooling the complexes through the phase transition, resulting in increased vesicle deformability. The rates of gel phase micellization, and of bilayer reformation from micelles at temperatures above T m, are decreased by 100-fold in P14A: DMPC complexes compared with with melittin: DMPC complexes. Freeze-fracture EM indicates that P14A suppresses the formation of the gel phase in DMPC bilayers at temperatures below T m. EM observations of the time-dependence of the reformation of bilayers from micelles after incubating P14A: DMPC micellar complexes at temperatures above T m indicate that micelles fuse to form growing bilayer sheets from which multilameliar vesicles eventually form. The presence of intramembranous particles (IP) on the fracture faces of both melittin: DMPC complexes and P14A: DMPC complexes in the fluid phase indicates that under the conditions of the study (50 mM Tris-HCl (pH 7.5), 5 mM EDTA) the peptides are organized as discrete aggregates that penetrate deeply into the bilayer.

Properties of lipid complexes with amphipathic helix-forming peptides: Role of distribution of peptide charges.

The peptides [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A are 18-residue peptides designed to form amphipathic helices with opposite charge distribution; [Glu1,8,Leu11,17] 18A having positively charged residues at the hydrophobic/hydrophilic interface. Both [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A strongly disrupt the bilayer structure as indicated by the relatively narrow lipid 1H and 31P NMR peaks. In addition, the 1H chemical shift of the quaternary ammonium methyl groups indicates that [Glu1,8,Leu11,17] 18A forms smaller lipoprotein particles with dimyristoylphosphatidylcholine (DMPC) than does [Glu4,9,Leu11,17] reverse-18A. However, motional properties of the lipid head group indicate that no specific salt bridges are formed between the phospholipid head group and the side chains of polar amino acids of either of the two peptides. In addition, the acyl chain conformation for the DMPC complexes with [Glu1,8,Leu11,17] 18A and with [Glu4,9,Leu11,17] reverse-18A are indistinguishable by the criterion of IR spectroscopy. The 2H linewidth of the solvent 2H2O remains narrower in frozen solutions of the DMPC-[Glu1,8,Leu11,17] 18A complexes suggesting the presence of more unfrozen bound water in this case. The two peptides exhibit many similarities in their interaction with lipids. However, [Glu1,8,Leu11,17] 18A can more readily lyse vesicles and activate lecithin:cholesterol acyltransferase. These differences do not appear to result from differences in specific charge interactions between the lipid and peptide but may be manifested through differences in hydration properties.

Studies of synthetic peptide analogs of the amphipathic helix. Structure of complexes with dimyristoyl phosphatidylcholine.

The amphipathic helix hypothesis for the lipid-associating domains of exchangeable plasma apolipoproteins has been further studied by analysis of the structure of the complexes formed between four synthetic peptide analogs of the amphipathic helix and dimyristoyl phosphatidylcholine (DMPC). Density gradient ultracentrifugation, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, 1H NMR, high sensitivity differential scanning calorimetry, and circular dichroism were the techniques used in these studies. The two analogs Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and 18A-Pro-18A whose sequences most strongly mimic native amphipathic sequences were found also most strongly to mimic apolipoprotein A-I in DMPC complex structure. The covalently linked dimer of the prototype amphipathic analog 18A, 18A-Pro-18A, appears to have greater lipid affinity than 18A. This presumably is the result of the cooperativity provided by two covalently linked lipid-associating domains in 18A-Pro-18A. The studies further suggest that the charge-reversed analog of the prototype 18A, reverse-18A, has the lowest lipid affinity of the four analogs studied and forms only marginally stable discoidal DMPC complexes. We postulate that this low lipid affinity is due predominantly, but not necessarily exclusively, to the lack of a hydrophobic contribution of lysine residues at the polar-nonpolar interface of reverse-18A versus 18A. The intermediate lipid affinity of des-Val10-18A, the fourth analog peptide, to produce a rank order of 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, supports this interpretation. Des-Val10-18A which has Val deleted from 18A has an amphipathic helical structure partially disrupted by the shift of 2 lysine residues away from the polar-nonpolar interface.

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: A study by oxygen quenching and fluorescence depolarization

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 °C as compared to that at 5 °C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC Complexes.

In vivo metabolism of human apoprotein A-I-phospholipid complexes. comparison with human high density lipoprotein-apoprotein A-I metabolism

The metabolism of apolipoprotein A-I was investigated in healthy young adults. The apoprotein was injected intravenously in one of the four following forms: radiolabelled high density lipoproteins, HDL incubated with labelled apo A-I, labelled A-I-dipalmitoyl- or dimyristoylphosphatidylcholine complexes or free apoprotein A-I. The radioactivity-time curves of high density lipoproteins were followed for 15 days. The kinetic studies demonstrate similar fractional catabolic rates, half-lifes and rates of synthesis. Fractional catabolic rate calculated from plasma data was in reasonable agreement with the value derived from the urine/plasma activity ratio. When free apo A-I was injected, the unusual form of the U/P ratio curve is interpreted as the result of different fates of the injected molecules. This study indicates that the half-life of apo A-I seems not to be affected by the form in which the apoprotein is injected and that kinetics of apo A-I-phospholipid complexes may be used as a valid index of apoprotein A-I/HDL metabolism.

Nanosecond rotational motions of apolipoprotein C-I in solution and in complexes with dimyristoylphosphatidylcholine.

Human apolipoprotein C-I (apo C-I) in solution, in monomeric and oligomeric form, and in micellar complexes with dimyristoylphosphatidylcholine (DMPC), below and above the phase transition temperature of DMPC, was investigated with steady-state and time-resolved fluorescence methods. The environment of the Trp residue of apo C-I, in each physical state, was evaluated from fluorescence spectra and their changes upon KI quenching. Rotational correlation times of Trp residues were obtained from fluorescence anisotropy decay measurements. Static fluorescence anisotropy was determined as a function of temperature for the Trp residues of apo C-I in all physical states and for diphenylhexatriene dissolved in apo C-I X DMPC complexes. It was found that the Trp residues of apo C-I in solution are exposed from 75 to 88% to the aqueous medium, depending on the state of self-association. On the other hand, the Trp residues in apo C-I X DMPC complexes are only 42-45% exposed to KI quenching through an environment distinct from water. Apolipoprotein C-I in all its physical forms had two rotational correlation times associated with Trp motions: a longer one dependent on the size and flexibility of the entire particle and a very short one in the range from 0.2 to 0.4 ns. The later correlation times correspond to local Trp residue motions. These Trp motions were not significantly affected by a transition from the gel to the liquid-crystalline state of the lipid in apo C-I X DMPC complexes, suggesting that there is no coupling between the local motions of lipids and those of Trp side chains of apo C-I.

Mechanism of lipid-protein interaction in the plasma lipoproteins: identification of a lipid-binding site in apolipoprotein A-II.

Apolipoprotein A-II (apoA-II) is a dimeric 77-residue apoprotein of human high-density lipoproteins. Previous studies indicate that residues 56--77 in the apoprotein do not bind phospholipid whereas residues 47--77 form a complex with dimyristoylphosphatidylcholine (DMPC). To further delineate the lipid-binding region between residues 47 and 77, we have prepared synthetic fragments of apoA-II corresponding to residues 54--77, 52--77, and 50--77 and have tested each fragment for its ability to interact with vesicles of DMPC. The interaction of the fragments was determined by changes in secondary structure as measured by circular dichroism and by isolation of peptide--DMPC complexes by ultracentrifugation in density gradients of KBr. By these criteria, only fragment 50--77 binds DMPC; there is an increase in alpha helicity from 17% to 41% when the fragment associates with lipid. Since the 56--77 fragment does not associate with phospholipid, we propose that the addition of residues Thr-Pro-Leu-Ile-Lys-Lys (corresponding to residues 50--55) to the 56--77 fragment gives the peptide the necessary sequence information for lipid binding. To further identify the important amino acid residues in the region of 50--55, we have substituted Leu-Ile with Ala-Ala. This substitution totally abolishes the lipid-binding capacity of the 50--77 fragment. On the other hand, substitution of Lys-Lys with Ser-Ser does not alter the lipid-binding capacity of the peptide. We conclude that residues 50--55 are important in lipid binding and that the hydrophobic center formed by Leu-Ile plays an important role.