Jere P. Segrest

A molecular theory of lipid—protein interactions in the plasma lipoproteins

Lipid-protein interactions are of fundamental importance in the structure of biological membranes and of plasma lipoproteins. From previous studies [1-3] it seems reasonable to assume that the interactions between phospholipids and lipoprotein-protein (apoprotein) constituents are fundamental to the binding of neutral lipid by the plasma lipoproteins. For example, there is an insignificant binding of cholesteryl ester by the apoproteins of human HDL * in the absence of phospholipids. We have recently presented studies describing the probable location of phospholipid-binding site(s) in the MN-glycoprotein of the human red cell membrane [4-6] and of plasma lipoproteins [7-12]. Phospholipid-binding regions do not appear to be uniformly distributed along the length of the polypeptide chain [7-11,13] ; certain fragments of apoLP-Ala, apoLP-Gln-I and apoLP-Gln-II preferentially bind phospholipid as corn-

[8] Single vertical spin density gradient ultracentrifugation

Publisher Summary Density gradient ultracentrifugation in the swing out rotor has been successfully applied to fractionation of the major lipoprotein species and their subspecies; however, it requires a minimum of 24–36 h to complete the separation of the major lipoprotein fractions in plasma. This chapter describes 13 separate vertical spin procedures for the preparative separation of a number of different plasma lipoproteins and a method for the quantitative analysis of lipoprotein cholesterol from plasma separated by single vertical spin (SVS) density gradient ultracentrifugation. The vertical rotor works on the principle that compression of the gradient geometry by the vertical rotor when at speed shortens spin time over more conventional ultracentrifugation techniques without loss of resolution. The single vertical spin procedure has a number of advantages over the more conventional methods including decreased spin time, decreased lipoprotein degradation, and good resolution. But there are certain disadvantages of this method also, such as wall adherence of VLDL and albumin, resolution, and plasma volume limit per rotor. However, the chapter addresses solutions to the problems of single vertical spin ultracentrifugation such as angled-head rotors.

Human erythrocyte membrane glycoprotein: A re-evaluation of the molecular weight as determined by SDS polyacrylamide gel electrophoresis

Abstract Molecular weights for the human erythrocyte membrane glycoprotein and other glycoproteins calculated relative to protein standards on SDS acrylamide gel electrophoresis depend upon the percent crosslinking of gels. This anomaly is due to a decreased binding of SDS to the oligosaccharide side chains relative to the polypeptide backbone; removal of sialic acid fails to correct the anomaly. Previous molecular weights proposed for the human erythrocyte membrane glycoprotein utilizing SDS gel electrophoresis are probably incorrect and a new value of 55,000 is proposed based on corrected SDS-gel data.

A DETAILED MOLECULAR BELT MODEL FOR APOLIPOPROTEIN A-I IN DISCOIDAL HIGH DENSITY LIPOPROTEIN

Apolipoprotein A-I (apoA-I) is the principal protein of high density lipoprotein particles (HDL). ApoA-I contains a globular N-terminal domain (residues 1–43) and a lipid-binding C-terminal domain (residues 44–243). Here we propose a detailed model for the smallest discoidal HDL, consisting of two apoA-I molecules wrapped beltwise around a small patch of bilayer containing 160 lipid molecules. The C-terminal domain of each monomer is ringlike, a curved, planar amphipathic α helix with an average of 3.67 residues per turn, and with the hydrophobic surface curved toward the lipids. We have explored all possible geometries for forming the dimer of stacked rings, subject to the hypothesis that the optimal geometry will maximize intermolecular salt bridge interactions. The resulting model is an antiparallel arrangement with an alignment matching that of the (nonplanar) crystal structure of lipid-free apoA-I.

The Amphipathic α Helix: A Multifunctional Structural Motif in Plasma Apolipoproteins

Publisher Summary The dominant structural motif of the peripheral apolipoproteins is the amphipathic helix, which is responsible for the reversible association of these proteins with lipids, as well as for many biological functions mediated by these apolipoproteins. This chapter reviews the different classes of amphipathic helices, using a combination of powerful computer programs to develop a comparison database and to analyze these structures. It also discusses their evolutionary origins, physical-chemical properties, X-ray structure determination, and conformational analysis. Although the structures of these lipoprotein classes are similar, they differ in relative proportion of lipids, in the apolipoprotein: lipid ratio and in the apolipoprotein species. The amphipathic α helix plays a pivotal role in the structure and functions of the exchangeable apolipoproteins. Site-directed mutagenesis and other molecular biology-based techniques are available for probing the structural motif. The location and properties of the amphipathic helices in apolipoproteins and the results are compared with recently developed and ever-expanding computer methods for the location and characterization. A variety of structure-function studies, including the activation of lipoprotein lipase, receptor recognition, lecithin-cholesterol acyltransferase (LCAT) activation, and antiviral and anti-inflammatory activities are also discussed.

Major glycoprotein of the human erythrocyte membrane: Evidence for an amphipathic molecular structure

Treatment of the major glycoprotein of the human red cell membrane [erythrocyte glycophorin (1)] with cyanogen bromide yields five fragments. On the basis of N-terminal analyses and tryptic overlaps three of these (designated C-1, C-2, and C-5) have been aligned as unique portions of a single polypeptide chain. C-5 and C-1 represent the N-terminal fragments, in that order, and the third, C-2, is the C-terminal fragment of the original polypeptide chain. Evidence is presented for a limited polypeptide microheterogeneity of the N-terminal portion of the molecule. From the amino acid composition and carbohydrate content of the three fragments, C-5, C-1, and C-2, the molecule can be divided into three distinct regions or domains. These are (a) a receptor or carbohydrate containing N-terminal segment, (b) an internal hydrophobic domain of approximately 30 residues, and (c) a hydrophilic, proline-rich C-terminal portion. This unique molecular topography suggests an amphipathic model for the in situ orientation of this molecule in which the hydrophobic domain of the glycoprotein lies within the hydrophobic interior of the membrane. This model is supported by lactoperoxidase-catalyzed 125I-labeling experiments which suggest that the N-terminal (receptor) half of the molecule (fragment C-1) is exposed to the external environment of the cell, while the C-terminal segment (fragment C-2) extends into the interior of the cell.

Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.

Clearance of excess cholesterol from cells by HDL is facilitated by the interaction of HDL apolipoproteins with cell-surface binding sites or receptors, a process that may be important in preventing atherosclerosis. In this study, synthetic peptides containing 18-mer amphipathic helices of the class found in HDL apolipoproteins (class A) were tested for their abilities to remove cholesterol and phospholipid from cultured sterol-laden fibroblasts and macrophages and to interact with cell-surface HDL binding sites. Lipid-free peptides containing two identical tandem repeats of class A amphipathic helices promoted cholesterol and phospholipid efflux from cells and depleted cellular cholesterol accessible for esterification by acyl CoA/cholesterol acyltransferase, similar to what was observed for purified apolipoprotein A-I. Peptide-mediated removal of plasma membrane cholesterol and depletion of acyl CoA/cholesterol acyltransferase-accessible cholesterol appeared to occur by separate mechanisms, as the latter process was less dependent on extracellular phospholipid. The dimeric amphipathic helical peptides also competed for high-affinity HDL binding sites on cholesterol-loaded fibroblasts and displayed saturable high-affinity binding to the cell surface. In contrast, peptides with a single helix had little or no ability to remove cellular cholesterol and phospholipid, or to interact with HDL binding sites, suggesting that cooperativity between two or more helical repeats is required for these activities. Thus, synthetic peptides comprising dimers of a structural motif common to exchangeable apolipoproteins can mimic apolipoprotein A-I in both binding to putative cell-surface receptors and clearing cholesterol from cells.

Only the Two End Helixes of Eight Tandem Amphipathic Helical Domains of Human Apo A-I Have Significant Lipid Affinity Implications for HDL Assembly

Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.

Membrane proteins: Amino acid sequence and membrane penetration

Abstract A computer study shows that the membrane-penetrating portion of the erythrocyte surface MN-glycoprotein (Winzler, 1969; Marchesi et al. , 1972) is distinguishable by informal cluster analysis from other segments of globular proteins when sequence length is plotted against hydrophobicity This analysis further suggests the possibility that other membrane-penetrating segments of proteins can be identified in the same way.

apoB-100 has a pentapartite structure composed of three amphipathic alpha-helical domains alternating with two amphipathic beta-strand domains. Detection by the computer program LOCATE.

Due to the great length of apolipoprotein (apo) B-100, the localization of lipid-associating domains in this protein has been difficult. To address this question, we developed a computer program called Locate that searches amino acid sequences to identify potential amphipathic alpha-helixes and beta-strands by using sets of rules for helix and strand termination. A series of model chimeric protein test datasets were created by tandem linking of amino acid sequences of multiple proteins containing four different secondary structural motifs: motif A (exchangeable plasma apolipoproteins); motif G (globular alpha-helical proteins); motif C (coiled-coil alpha-helical proteins); and motif B (beta pleated-sheet proteins). These four test datasets, as well as randomly scrambled sequences of each dataset, were analyzed by Locate using increasingly stringent parameters. Using intermediately stringent parameters under which significant numbers of amphipathic helixes were found only in the unscrambled motif A, two dense clusters of putative lipid-associating amphipathic helixes were located precisely in the middle and at the C-terminal end of apoB-100 (a sparse cluster of class G* helixes is located at the N-terminus). The dense clusters are located between residues 2103 through 2560 and 4061 through 4338 and have densities of 2.4 and 2.2 amphipathic helixes per 100 residues, respectively; under these conditions, motif A has a density of 1.4 amphipathic helixes per 100 residues. These two domains correspond closely to the two major apoB-100 lipid-associated domains at residues 2100 through 2700 and 4100 through 4500 using the principle of releasability of tryptic peptides from trypsin-treated intact low-density lipoprotein. The classes of amphipathic helixes identified within these two putative lipid-associating domains are considerably more diverse than those found in the exchangeable plasma apolipoproteins. Interestingly, apoB-48 terminates at the N-terminal edge of the middle cluster. By using a similar strategy for analysis of amphipathic beta-strands, we discovered that the two gap regions between the three amphipathic helix clusters are highly enriched in putative amphipathic beta-strands, while the three amphipathic helical domains are essentially devoid of this putative lipid-associating motif. We propose, therefore, that apoB-100 has a pentapartite structure, NH2-alpha 1-beta 1-alpha 2-beta 2-alpha 3-COOH, with alpha 1 representing a globular domain.