Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: evaluation of reversible unfolding conditions

Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium salinarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles. To study the effect on protein stability by substitutions on the hydrophobic surface with polar residues, the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)]. sbR(Q2) was more sensitive to SDS-induced unfolding than sbR(WT) under equilibrium conditions, and kinetic experiments showed that sbR(Q2) was more sensitive to acid-induced denaturation and thermal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the detergent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are important for the stability of sbR. Our experiments provide the basis for studying the thermodynamic stability of sbR by evaluating reversible folding and unfolding conditions in DMPC/CHAPSO/SDS mixed micelles.     

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.     

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.     

Nuclear magnetic resonance investigation of the interactions with phospholipid of an amphipathic alpha-helix-forming peptide of the apolipoprotein class

To further understand the packing of amphipathic alpha-helices of apolipoproteins in serum lipoproteins, we have investigated the interactions with dimyristoyl phosphatidylcholine (DMPC) of a 13C-labeled, 18-residue peptide (18A) which can form an amphipathic alpha-helix. This peptide whose amino acid sequence is DWLKAFYDKVAEKLKEAF has the positive-negative residue clustering typical of the apolipoprotein class of amphipathic helix. 13CH3-alanine was introduced as the 11th residue of 18A so that the 13CH3 group protrudes on the apolar side of the amphipathic helix. [13C]NMR spectra of [13C-Ala11]18A in discoidal complexes with DMPC show three resonances from the Ala-13CH3 group; one originates from 18A in aqueous solution, while those at chemical shifts (delta) of 15.2 and 16.4 ppm are assigned to 18A in the "edge" and "faces," respectively, of the discoidal complex. The proportion of 18A in the faces of the discoidal complex increases as the size of the disk is increased by raising the lipid/peptide ratio. 18A covers the edge of the disk so that the 13CH3-Ala side chain from these molecules is in contact with DMPC acyl chains. [13C-Ala11]18A bound to the surface of an egg PC small unilamellar vesicle gives a single resonance from 18A at delta 16.3 ppm consistent with there being no edge location. Cooling 18A-DMPC disks to 15 degrees C crystallizes the DMPC bilayer and restricts the motion of the 13CH3-Ala group of the 18A molecules. The molecular motions of the side chains of the amphipathic helix are sensitive to their location in the disk and to PC molecular packing.     

Effects of protein oxidation on the structure and stability of model discoidal high-density lipoproteins

High-density lipoproteins (HDLs) prevent atherosclerosis by removing cholesterol from macrophages and by providing antioxidants for low-density lipoproteins. Oxidation of HDLs affects their functions via the complex mechanisms that involve multiple protein and lipid modifications. To differentiate between the roles of oxidative modifications in HDL proteins and lipids, we analyzed the effects of selective protein oxidation by hypochlorite (HOCl) on the structure, stability, and remodeling of discoidal HDLs reconstituted from human apolipoproteins (A-I, A-II, or C-I) and phosphatidylcholines. Gel electrophoresis and electron microscopy revealed that, at ambient temperatures, protein oxidation in discoidal complexes promotes their remodeling into larger and smaller particles. Thermal denaturation monitored by far-UV circular dichroism and light scattering in melting and kinetic experiments shows that protein oxidation destabilizes discoidal lipoproteins and accelerates protein unfolding, dissociation, and lipoprotein fusion. This is likely due to the reduced affinity of the protein for lipid resulting from oxidation of Met and aromatic residues in the lipid-binding faces of amphipathic alpha-helices and to apolipoprotein cross-linking into dimers and trimers on the particle surface. We conclude that protein oxidation destabilizes HDL disk assembly and accelerates its remodeling and fusion. This result, which is not limited to model discoidal but also extends to plasma spherical HDL, helps explain the complex effects of oxidation on plasma lipoproteins.     

Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.     

Conformation and lipid binding of the N-terminal (1-44) domain of human apolipoprotein A-I

Because of its role in reverse cholesterol transport, human apolipoprotein A-I is the most widely studied exchangeable apolipoprotein. Residues 1-43 of human apoA-I, encoded by exon 3 of the gene, are highly conserved and less well understood than residues 44-243, encoded by exon 4. In contrast to residues 44-243, residues 1-43 do not contain the 22 amino acid tandem repeats thought to form lipid binding amphipathic helices. To understand the structural and functional roles of the N-terminal region, we studied a synthetic peptide representing the first 44 residues of human apoA-I ([1-44]apoA-I). Far-ultraviolet circular dichroism spectra showed that [1-44]apoA-I is unfolded in aqueous solution. However, in the presence of n-octyl beta-d-glucopyranoside, a nonionic lipid mimicking detergent, above its critical micelle concentration ( approximately 0.7% at 25 degrees C), sodium dodecyl sulfate, an ionic detergent, above its CMC ( approximately 0.2%), trimethylamine N-oxide, a folding inducing organic osmolyte, or trifluoroethanol, an alpha-helix inducer, alpha-helical structure was formed in [1-44]apoA-I up to approximately 45%. Characterization by density gradient ultracentrifugation and visualization by negative staining electron microscopy demonstrated that [1-44]apoA-I interacts with dimyristoylphosphatidylcholine (DMPC) over a wide range of lipid:peptide ratios from 1:1 to 12:1 (w/w). At 1:1 DMPC:[1-44]apoA-I (w/w) ratio, discoidal complexes with composition approximately 4:1 (w/w) and approximately 100 A diameter were formed in equilibrium with free peptide. At higher ratios, discoidal complexes were shown to exist together with a heterogeneous population of lipid vesicles with peptide bound also in equilibrium with free peptide. When bound to DMPC, [1-44]apoA-I has approximately 60% helical structure, independent of whether it forms discoidal or vesicular complexes. This helical content is consistent with that of the predicted G helix (residues 8-33). Our data provide the first strong and direct evidence that the N-terminal region of apoA-I binds lipid and can form discoidal structures and a heterogeneous population of vesicles. In doing so, approximately 60% of this region folds into alpha-helix from random coil. The composition of the 100 A discoidal complex is approximately 5 [1-44]apoA-I and approximately 150 DMPC molecules per disk. The helix length of 5 [1-44]apoA-I molecules in lipid-bound form is just long enough to wrap around the DMPC bilayer disk once.     

Conformation and lipid binding of a C-terminal (198-243) peptide of human apolipoprotein A-I

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 approximately 60% alpha-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 and 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 approximately 50% alpha-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, approximately 60% alpha-helix was induced in the peptide. In contrast, SDS, an anionic lipid-mimicking detergent, induced helical folding in the peptide at a concentration of approximately 0.003% (approximately 100 microM), approximately 70-fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solubilize dimyristoylphosphatidylcholine (DMPC) liposomes and fold into approximately 60% alpha-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy demonstrated that the peptide binds to DMPC with a high affinity to form at least two sizes of relatively homogeneous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide weight ratio, diameter, and density) of both complexes are similar to those of plasma A-I/DMPC complexes formed under similar conditions: small discoidal complexes (approximately 3:1 weight ratio, approximately 110 A, and approximately 1.10 g/cm3) formed at an initial 1:1 weight ratio and larger discoidal complexes (approximately 4.6:1 weight ratio, approximately 165 A, and approximately 1.085 g/cm3) formed at initial 4:1 weight ratio. The cross-linking data for the peptide on the complexes of two sizes is consistent with the calculated peptide numbers per particle. Compared to the approximately 100 A disk-like complex formed by the N-terminal peptide in which helical structure was insufficient to cover the disk edge by a single belt, the compositions of these two types of complexes formed by the C-terminal peptide are more consistent with a "double belt" model, similar to that proposed for full-length apoA-I. 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 C-terminal peptide model can mimic the interaction with the phospholipid of plasma apoA-I to form two sizes of homogeneous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.     

The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles.

The details of how high density lipoprotein (HDL) microstructure affects the conformation and net charge of apolipoprotein (apo) A-I in various classes of HDL particles have been investigated in homogeneous recombinant HDL (rHDL) particles containing apoA-I, palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate. Isothermal denaturation with guanidine HCl was used to monitor alpha-helix structural stability, whereas electrokinetic analyses and circular dichroism were used to determine particle charge and apoA-I secondary structure, respectively. Electrokinetic analyses show that at pH 8.6 apoA-I has a net negative charge on discoidal (POPC.apoA-I) particles (-5.2 electronic units/mol of apoA-I) which is significantly greater than that of apoA-I either free in solution or on spherical (POPC.cholesteryl oleate.apoA-I) rHDL (approximately -3.5 electronic units). Raising the POPC content (32-128 mol/ml of apoA-I) of discoidal particles 1) increases the particle major diameter from 9.3 to 12.1 nm, 2) increases the alpha-helix content from 62 to 77%, and 3) stabilizes the helical segments by increasing the free energy of unfolding (delta GD degree) from 1.4 to 3.0 kcal/mol of apoA-I. Raising the POPC content (28-58 mol/mol of apoA-I) of spherical particles 1) increases the particle diameter from 7.4 to 12.6 nm, 2) increases the percent alpha-helix from 62 to 69%, and 3) has no significant effect on delta GD degree (2.2 kcal/mol of apoA-I). This study shows that different HDL subspecies maintain particular apoA-I conformations that confer unique charge and structural characteristics on the particles. It is likely that the charge and conformation of apoA-I are critical molecular properties that modulate the metabolism of HDL particles and influence their role in cholesterol transport.     

Kinetic stabilization and fusion of apolipoprotein A-2:DMPC disks: comparison with apoA-1 and apoC-1

Denaturation studies of high-density lipoproteins (HDL) containing human apolipoprotein A-2 (apoA-2) and dimyristoyl phosphatidylcholine indicate kinetic stabilization. Circular dichroism (CD) and light-scattering melting curves show hysteresis and scan rate dependence, indicating thermodynamically irreversible transition with high activation energy E(a). CD and light-scattering data suggest that protein unfolding triggers HDL fusion. Electron microscopy, gel electrophoresis, and differential scanning calorimetry show that such fusion involves lipid vesicle formation and dissociation of monomolecular lipid-poor protein. Arrhenius analysis reveals two kinetic phases, a slower phase with E(a,slow) = 60 kcal/mol and a faster phase with E(a,fast) = 22 kcal/mol. Only the fast phase is observed upon repetitive heating, suggesting that lipid-poor protein and protein-containing vesicles have lower kinetic stability than the disks. Comparison of the unfolding rates and the melting data recorded by differential scanning calorimetry, CD, and light scattering indicates the rank order for the kinetic disk stability, apoA-1 > apoA-2 > apoC-1, that correlates with protein size rather than hydrophobicity. This contrasts with the tighter association of apoA-2 than apoA-1 with mature HDL, suggesting different molecular determinants for stabilization of model discoidal and plasma spherical HDL. Different effects of apoA-2 and apoA-1 on HDL fusion and stability may reflect different metabolic properties of apoA-2 and/or apoA-1-containing HDL.     

Interaction of human plasma high-density lipoprotein HDL2b with discoidal complexes of dimyristoylphosphatidylcholine and apolipoprotein A-I

The interaction of HDL2b, a major subclass (d = 1.063 - 1.100 g/ml) of human plasma high-density lipoproteins, with discoidal complexes composed of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (weight ratio, DMPC/apolipoprotein A-I (2.1 - 2.5:1); dimensions, 10.0 x 4.4 nm) was investigated. Incubation at 37 degrees C for 4.5 h of HDL2b with discoidal complexes resulted in a transfer of DMPC from the discoidal complexes to the HDL2b, a release of lipid-free apolipoprotein A-I from the discoidal complexes during such transfer, and a dissociation of some apolipoprotein A-I from the HDL2b surface. The number of discoidal complexes degraded during interaction with HDL2b depended on the initial molar ratio of HDL2b to discoidal complexes. Approximately one molecule of HDL2b was required for the degradation of one discoidal complex particle, and the degradation process appeared limited by the capacity of the HDL2b for uptake of DMPC. Degradation of discoidal complexes was also observed when human plasma LDL (d = 1.006-1.063 g/ml) was substituted for HDL2b in the interaction mixture.     

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.     

Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology

Human apolipoprotein C-I (apoC-I) is an exchangeable apolipoprotein that binds to lipoprotein particles in vivo. In this study, we employed a LC-MS/MS assay to demonstrate that residues 38-51 of apoC-I are significantly protected from proteolysis in the presence of 1,2-dimyristoyl-3-sn-glycero-phosphocholine (DMPC). This suggests that the key lipid-binding determinants of apoC-I are located in the C-terminal region, which includes F42 and F46. To test this, we generated site-directed mutants substituting F42 and F46 for glycine or alanine. In contrast to wild-type apoC-I (WT), which binds DMPC vesicles with an apparent Kd [Kd(app)] of 0.89 microM, apoC-I(F42A) and apoC-I(F46A) possess 2-fold weaker affinities for DMPC with Kd(app) of 1.52 microM and 1.58 microM, respectively. However, apoC-I(F46G), apoC-I(F42A/F46A), apoC-I(F42G), and apoC-I(F42G/F46G) bind significantly weaker to DMPC with Kd(app) of 2.24 microM, 3.07 microM, 4.24 microM, and 10.1 microM, respectively. Sedimentation velocity studies subsequently show that the protein/DMPC complexes formed by these apoC-I mutants sediment at 6.5S, 6.7S, 6.5S, and 8.0S, respectively. This is compared with 5.0S for WT apoC-I, suggesting the shape of the particles was different. Transmission electron microscopy confirmed this assertion, demonstrating that WT forms discoidal complexes with a length-to-width ratio of 2.57, compared with 1.92, 2.01, 2.16, and 1.75 for apoC-I(F42G), apoC-I(F46G), apoC-I(F42A/F46A), and apoC-I(F42G/F46G), respectively. Our study demonstrates that the C-terminal amphipathic alpha-helix of human apoC-I contains the major lipid-binding determinants, including important aromatic residues F42 and F46, which we show play a critical role in stabilizing the structure of apoC-I, mediating phospholipid interactions, and promoting discoidal particle morphology.     

Direct Measurement of the Structure of Reconstituted High-Density Lipoproteins by Cryo-EM

Early forms of high-density lipoproteins (HDL), nascent HDL, are formed by the interaction of apolipoprotein AI with macrophage and hepatic ATP-binding cassette transporter member 1. Various plasma activities convert nascent to mature HDL, comprising phosphatidylcholine (PC) and cholesterol, which are selectively removed by hepatic receptors. This process is important in reducing the cholesterol burden of arterial wall macrophages, an important cell type in all stages of atherosclerosis. Interaction of apolipoprotein AI with dimyristoyl (DM)PC forms reconstituted (r)HDL, which is a good model of nascent HDL. rHDL have been used as an antiathersclerosis therapy that enhances reverse cholesterol transport in humans and animal models. Thus, identification of the structure of rHDL would inform about that of nascent HDL and how rHDL improves reverse cholesterol transport in an atheroprotective way. Early studies of rHDL suggested a discoidal structure, which included pairs of antiparallel helices of apolipoprotein AI circumscribing a phospholipid bilayer. Another rHDL model based on small angle neutron scattering supported a double superhelical structure. Herein, we report a cryo-electron microscopy-based model of a large rHDL formed spontaneously from apolipoprotein AI, cholesterol, and excess DMPC and isolated to near homogeneity. After reconstruction we obtained an rHDL structure comprising DMPC, cholesterol, and apolipoprotein AI (423:74:1 mol/mol) forming a discoidal particle 360 Å in diameter and 45 Å thick; these dimensions are consistent with the stoichiometry of the particles. Given that cryo-electron microscopy directly observes projections of individual rHDL particles in different orientations, we can unambiguously state that rHDL particles are protein bounded discoidal bilayers.     

Interaction of tryptic peptides of apolipoprotein B-100 with dimyristoylphosphatidylcholine

Apolipoprotein B-100, the major protein constituent of human plasma low-density lipoproteins (LDL), was carboxyamidomethylated, digested with trypsin and the water-soluble tryptic peptides were coincubated with liposomes of dimyristoylphosphatidylcholine (DMPC). At 24.3 degrees C the peptides induced lipid solubilization as evidenced by optical clearing of the lipid-peptide mixture. Lipid-peptide complexes were isolated by density-gradient ultracentrifugation in KBr and had the following properties: DMPC/peptide ratio of 5.6 (w/w); buoyant density of 1.07-1.09 g/ml; discoidal morphology (51 +/- 4 X 260 +/- 28 A) as determined by electron microscopy; and molecular weight of 1.5 X 10(6) as determined by nondenaturing polyacrylamide gel electrophoresis. Compared to liposomes and sonicated vesicles of DMPC, the lipid-peptide complexes had a more rigid structure as assessed by fluorescence polarization. Whereas intact LDL had 42% alpha-helix and 15% beta-pleated sheet, the lipid-peptide complexes contained 70% alpha-helix and less than 5% beta-pleated sheet. The lipid-peptide complexes did not bind to the fibroblast high-affinity LDL receptor. These results show that specific regions in apolipoprotein B-100 which interact with phospholipid have an amphipathic character and may represent primary sites for lipid-protein interaction in LDL.     

Effect of phosphatidylethanolamine on the properties of phospholipid-apolipoprotein complexes

Plasma high density lipoproteins (HDL) are synthesized in intestinal mucosal cells and hepatocytes and are secreted into the blood. Factors influencing the structure and function of these HDL, such as lipid and protein composition, are poorly understood. It appears, however, that intracellular, discoidal HDL are enriched, relative to plasma HDL, in phosphatidylethanolamine (PE), a phospholipid known to generate unusual, nonbilayer structures of putative physiological significance. Although incubation of dimyristoylphosphatidylcholine (DMPC) with apolipoprotein A-I at the gel-liquid crystalline phase transition temperature results in the spontaneous formation of lipid-protein complexes, the presence of proportionately small amounts of PE prevents the formation of such complexes, suggesting that PE profoundly alters the phase properties of the phospholipid bilayers. However, by using a detergent-mediated method for the formation of PE-rich model nascent HDL from phospholipids and apolipoprotein A-I, lipid-protein complexes containing as much as 75% DLPE could be formed, thus demonstrating that the presence of PE causes a kinetic, rather than a thermodynamic, barrier to spontaneous complex formation. The products contained a DLPE:DMPC molar ratio similar to that of the initial incubation mixture; however, as the mole percentage of DLPE increased, the products became less heterogeneous, the buoyant density of the products increased, and the Stokes diameter of the products decreased. Similar results were obtained when dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE) were employed in lieu of DLPE. Electron microscopy of complexes containing DLPE and DMPC at a 1:1 molar ratio showed that these particles possessed a discoidal, bilayer morphology similar to that seen with complexes containing only phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of dissociation of human apolipoprotein A-I from complexes with dimyristoylphosphatidylcholine as studied by guanidine hydrochloride denaturation

The reversibility of the binding of human apolipoprotein A-I (apo A-I) to phospholipid has been monitored through the influence of guanidine hydrochloride (Gdn-HCl) on the isothermal denaturation and renaturation of apo A-1/dimyristoylphosphatidylcholine (DMPC) complexes at 24 degree C. Denaturation was studied by incubating discoidal 1:100 and vesicular 1:500 mol/mol apo A-I/DMPC complexes with up to 7 M Gdn-HCl for up to 72 h. Unfolding of apo A-I molecules was observed from circular dichroism spectra while the distribution of protein between free and lipid-associated states was monitored by density gradient ultracentrifugation. The ability of apo A-I to combine with DMPC in the presence of Gdn-HCl at 24 degrees C was also investigated by similar procedures. In both the denaturation and renaturation of 1:100 and 1:500 complexes, the final values of the molar ellipticity and the ratio of free to bound apo A-I at various concentrations of Gdn-HCl are dependent on the initial state of the lipid and protein; apo A-I is more resistant to denaturation when Gdn-HCl is added to existing complexes than to a mixture of apo A-I and DMPC. There is an intermediate state in the denaturation pathway of apo A-I/DMPC complexes which is not present in the renaturation; the intermediate comprises partially unfold apo A-I molecules still associated with the complex by some of their apolar residues. Complete unfolding of the alpha helix and subsequent desorption of the apo A-I molecules from the lipid/water interface involve cooperative exposure of these apolar residues to the aqueous phase. The energy barrier associated with this desorption step makes the binding of apo A-I to DMPC a thermodynamically irreversible process. Consequently, binding constants of apo A-I and PC cannot be calculated simply from equilibrium thermodynamic treatments of the partitioning of protein between free and bound states. Apo A-I molecules do not exchange freely between the lipid-free and lipid-bound states, and extra work is required to drive protein molecules off the surface. The required increased in surface pressure can be achieved by a net mass transfer of protein to the surface; in vivo, increases in the surface pressure of lipoproteins by lipolysis can cause protein desorption.     

Human apolipoprotein A-I forms thermally stable complexes with anionic but not with zwitterionic phospholipids

The mechanism of the association of human plasma apolipoprotein A-I (apo A-I) with the acidic phospholipids, dimyristoylphosphatidylglycerol (DMPG), egg yolk phosphatidylglycerol, and dioleoylphosphatidylserine as well as with the zwitterionic dimyristoylphosphatidylcholine (DMPC) has been studied using turbidimetry, circular dichroism, high-sensitivity differential scanning calorimetry, and electron microscopy. The association of apo A-I with multilamellar liposomes of acidic phospholipids is rapid over a broad temperature range at and above the temperature of the lipid gel to liquid crystalline transition, Tc. This is in contrast to zwitterionic phosphatidylcholine which recombines with apo A-I only over a narrow temperature range around Tc. The complex of apo A-I with DMPC denatures at elevated temperatures giving rise to a calorimetrically detectable transition. The temperature range and width of this transition is shown to be markedly dependent on the heating rate. This is again in contrast to apo A-I recombinants with DMPG which show no calorimetrically detectable thermal denaturation, at least in a temperature range up to 100 degrees C. Also circular dichroism data indicate high resistance of apo A-I to thermal unfolding in the presence of DMPG. It is concluded that the complexes of apo A-I with DMPC are thermodynamically stable only at temperatures near Tc, whereas above and below this temperature range the stability of these recombinants is determined by kinetic factors. In contrast, complexes of apo A-I with DMPG and other acidic phospholipids may be thermodynamically stable over a wide temperature range greater than or equal to Tc. In spite of these fundamental differences between zwitterionic and acidic phospholipids in their mode of association with apo A-I, the binding affinity and the morphology of the recombinants are similar. Both apo A-I X DMPC and apo A-I X DMPG complexes form lipoprotein particles having a discoidal shape.     

Hydrophobic Core Mutations Associated with Cataract Development in Mice Destabilize Human ��D-Crystallin

The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human γD-crystallin (HγD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key β-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the γ-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract in mice, presumably an aggregated state of the mutant crystallins. It has not been possible to identify the aggregating precursor within lens tissues. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of γ-crystallins, mouse mutant substitutions were introduced into HγD-Crys. The mutant proteins L5S, V75D, and I90F were expressed and purified from Escherichia coli. WT HγD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded and the C-td native-like. L5S and V75D in the N-td also displayed three-state unfolding transitions, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. I90F destabilized the C-td, shifting the overall unfolding transition to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments showed that the N-tds of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded more rapidly. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.     

Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide.

To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine-N-oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C-1 (apoC-1, 57 residues) containing point mutations in the predicted alpha-helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild-type and plasma apoC-1 in neutral low-salt solutions superimpose, indicating 31 +/- 4% alpha-helical structure at 22 degrees C that melts reversibly with T(m,WT) = 50 +/- 2 degrees C and van't Hoff enthalpy deltaH(v,WT)(Tm) = 18 +/- 2 kcal/mol. G15A substitution leads to an increased alpha-helical content of 42 +/- 4% and an increased T(m,G15A) = 57 +/- 2 degrees C, which corresponds to stabilization by delta deltaG(app) = +0.4 +/- 1.5 kcal/mol. G15P mutant has approximately 20% alpha-helical content at 22 degrees C and unfolds with low cooperativity upon heating to 90 degrees C. R23P and T45P mutants are fully unfolded at 0-90 degrees C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative alpha-helical unit in apoC-1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self-associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil-to-helix transition in apoC-1, with the maximal alpha-helical content reaching 74%. Comparison with the maximal alpha-helical content of 73% observed in lipid-bound apoC-1 suggests that the TMAO-stabilized secondary structure resembles the functional lipid-bound apolipoprotein conformation.