Abstract
Four peptides capable of forming an amphipathic alpha-helix have been synthesized and their conformational and lipid-binding properties studied. These peptides have been designed to vary the alpha-helix-forming potential as well as the charge distribution of the model peptide. The resulting peptide analogs and their complexes with dimyristoyl phosphatidylcholine were studied by using right angle light scattering, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry techniques. The four analogs, [Glu4,9, Leu11,17] (reverse-18A, [Glu4,9, Leu5,11,17] reverse-18A, [Glu1,8, Leu11,17] 18A, and [Glu1,8, Leu5,11,17] 18A were derived from a model amphipathic peptide Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) whose lipid-associating properties strongly mimic apolipoprotein A-I or derived from Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe (reverse-18A), a peptide with little affinity for lipid and having a reversed charge distribution compared to the 18A peptide. We have shown that by substituting glutamic acid and leucine for aspartic acid and alanine, respectively, in a weak lipid-associating amphipathic helix peptide, the lipid-associating ability can be increased. Thus, peptides with both kinds of charge distribution can associate with the lipid. The ability of the peptide to disrupt phospholipid bilayers, however, is higher for 18A analogs compared to the reverse-18A analogs even after increasing the helix-forming potential and hydrophobicity. In addition to forming smaller lipoprotein particles, the modified 18A analogs were much superior to the modified reverse-18A analogs in their ability to activate the enzyme lecithin:cholesterol acyltransferase. This demonstrates that the positions of charged residues in the amphipathic helix play an important role in lecithin:cholesterol acyltransferase activation.
Highlights
Four peptides capable of forming an amphipathic a- lipoproteins [1]
Ala-Lys-Glu-Leu-Glu-Lys-Ala-Ph(ereverse-lSA), a In our previous studies [2,3,4,5,6], peptide analogs of the ampeptide with little affinity for lipid and having a re- phipathic helix were synthesized with differences in the disversed charge distribution compared to the 18A pep- tribution of the charged residues
Inthe original model of the amphipathic helix-lipid interaction, it was proposed that there were specific electrostatic interactionsbetween the charged groups on the peptide and on the lipid [1].In the present work we demonstrate that some amphipathic helices with both "normal" as well as
Summary
Electron Microscopy-Prepared peptide-DMPC complexes were stained with 2% (w/v) potassium phosphotungstate, pH 5.9, and examined with a PhilipsEM 400 microscope on carbon-coated Formvar grids. Intrinsic Tryptophan Fluorescence-Fluorescence spectra of both peptide and peptide-DMPC complexes were recorded a t room temperature with excitation at 287 nm using a SPF-500 spectrofluorimeter. Circular Dichroism-CD spectra were obtained with an Aviv model. The instrument is equipped with a 50 kHz photoelastic modulator and an end-on photomultiplier. The instrument was calibrated with d-10-camphorsulfonic acid [21]. The concentration of peptide solutions was determined by amino acid analysis. 18A, Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-The abbreviations used are: DMPC, dimyristoyl phosphatidylcho-. Leu-Lys-Glu-Ala-Phe; reverse-MA, Lys-Trp-Leu-Asp-Ala-Phe-Tyr-line; DSC, differential scanning calorimetry; apoA-I, apolipoprotein
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