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In a recent classification of biologically active amphipathic alpha-helixes, the lipid-associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J.P., De Loof, H., Dohlman, J.G., Brouillette, C.G., Anantharamaiah, G.M. Proteins 8:103-117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the alpha-amino terminus and an amide at the alpha-carboxyl terminus, to obtain Ac-18A-NH2, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac-18A-NH2, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end-groups of 18A stabilizes the alpha-helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self-association of the blocked peptide in aqueous solution. The free energy of binding to the PC-water interface is increased only by about 3% (from -8.0 kcal/mol for 18A to -8.3 kcal/mol for Ac-18A-NH2). The Ac-18A-NH2 has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac-18A-NH2 more closely resembles the behavior of the apolipoprotein A-I, which is the major protein component of high-density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac-18A-NH2 peptide is greater than the 18A analog and comparable to that observed with the apo A-I. In the case of Ac-18A-NH2, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles.

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Apolipoprotein A-I (apoA-I), the major protein component of serum high-density lipoproteins (HDL), was found to inhibit herpes simplex virus (HSV)-induced cell fusion at physiological (approximately 1 microM) concentrations, whereas HDL did not exert any inhibitory effect. Lipid-associating, synthetic amphipathic peptides corresponding to residues 1-33 (apoA-I[1-33]) or residues 66-120 (apoA-I[66-120]) of apoA-I, also inhibited HSV-induced cell fusion, whereas a peptide corresponding to residues 8-33 of apoA-I (apoA-I[8-33]), which fails to associate with lipids, did not exert any inhibitory effect. These results suggest that lipid binding may be a prerequisite for peptide-mediated fusion inhibition. Consistent with this idea, a series of lipid-binding 22-amino-acid-residue-long synthetic amphipathic peptides that correspond to the amphipathic helical domains of apoA-I (A-I consensus series), or 18-residue-long model amphipathic peptides (18A series), were found to exert variable levels of fusion-inhibitory activity. The extent of fusion-inhibitory activity did not correlate with hydrophobic moment, hydrophobicity of the nonpolar face, helix-forming ability, or lipid affinity of the different peptides. Peptides in which the nonpolar face was not interrupted by a charged residue displayed greater fusion-inhibitory activity. Also, the presence of positively charged residues at the polar-nonpolar interface was found to correlate with higher fusion-inhibitory activity.

The major protein of high density llpoproteln (HDL), apolipoprotein (apo) A-l, is the major activator of the plasma enzyme lecithin:cholesterol acyltransferase (LCAT). A consensus amlno acid sequence has been defined for the eight, 22-resldue long, tandem amphlpathic helical repeats located in the carboxy-terminal region of apo A-l. A series of 22 and 44mer synthetic peptide analogues of the consensus domain, differing only In their 13th amlno acid residue, were prepared and tested for LCAT activation. One of the peptldes was found to equal apo A-l In LCAT activation. This is the first time a peptide activator for LCAT that rivals the activity of apo A-l in the vesicular and discoldal egg phosphatidylcholine assay systems has been synthesized. Based on these results, we propose that the major LCAT-actlvating domain of apo A-l resides in the 22mer tandem repeats, each containing Glu at the 13th residue and located between residues 66 and 121 In the native apolipoprotein. (Arteriosclerosis 10:95-105, January/February 1990)

The molecular mechanism of helix nucleation in peptides and proteins is not yet understood and the question of whether sharp turns in the polypeptide backbone serve as nuclei for protein folding has evoked controversy. A recent study of the conformation of a tetrapeptide containing the stereochemically constrained residue alpha-aminoisobutyric acid, both in solution and the solid state, yielded a structure consisting of two consecutive beta-turns, leading to an incipient 3(10) helical conformation. :This led us to speculate that specific tri- and tetrapeptide sequences may indeed provide a helical twist to the amino-terminal segment of helical regions in proteins and provide a nucleation site for further propagation. The transformation from a 3(10) helical structure to an alpha-helix should be facile and requires only small changes in the phi and psi conformational angles and a rearrangement of the hydrogen bonding pattern. If such a mechanism is involved then it should be possible to isolate an incipient 3(10) helical conformation in a tripeptide amide or tetrapeptide sequence, based purely on the driving force derived from short-range interactions. We have synthesised and studied the model peptide pivaloyl-Pro-Pro-Ala-NHMe (compound I) and provide here spectroscopic evidence for a 3(10) helical conformation in compound I.

Peptide analogues of the class A amphipathic helixes from exchangeable apolipoproteins mimic apolipoprotein (apo) A-I in a number of ways, including the ability to activate the enzyme lecithin: cholesterol acyltransferase, to associate with high density lipoproteins (HDLs), and to form HDL-like particles in the presence of lipids. This study investigated the metabolic properties of several of these peptide analogues in the rat Peptide analogues studied were 18A (referred to as L-18A to differentiate it from D-18A, and which mimics apolipoprotein amphipathic helical domains in its charge distribution), 37pA (a dimer of two 18A monomers separated by a proline), 18R (with reversed charge distribution compared with 18A), and D-18A (identical in amino acid sequence to 18A but synthesized from D-amino acids). Peptides were radiolabeled with l2S l. In addition, metabolism of rat and human 1Z5 I-apo A-I and human 14 C-apo A-I was studied; no significant differences in clearance of these preparations were seen. Clearance data were fitted to multiexponential equations to give half-times of clearance; biexponential equations consistently provided the best nonlinear least-squares curve fit The order of relative lipid affinity determined in vitro was 37pA>apo A-I>D-18A=L-18A>18R. Half-times of clearance were in the same approximate rank order: 37pA, 6.9±3J hours (mean±SD); apo A-I, 6.9±1.8 hours; D-18A, 4.0 ±1.0 hours; L-18A, 4.6 ±1.6 hours; and 18R, 0.9±0.1 hour. Rats injected with L-18A had five times more radioactivity in the urine than did rats injected with D-18A. All urine radioactivity was present as free I23 I in rats injected with L-18A or apo A-I but was present as intact peptide (with no free II5 I) in rats injected with D-18A. The majority of radioactivity in L-and D-18A-injected rats was associated with the thyroid gland (in the case of L-18A), the liver, and the kidney. In summary, the rates of clearance of amphipathic helical peptides from the plasma compartment in rats decrease as the affinities of the peptides for lipoprotein surfaces increase. Stereoconformation did not affect the rate of clearance of peptide analogues. Although a significant proportion of radioactivity in L and D-18A-injected animals was associated with the kidney, excretion of intact peptides in the urine did not appear to be a major route of clearance. (