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Abstract
Apolipoprotein (apo) B-100, the protein component of the blood serum low density lipoproteins (LDL), incorporates four proline-rich clusters within its amino-acid residues sequence. These structural domains have been proposed to have the potential to form anionic amphiphilic $\beta$-sheets, and to contribute to the protein binding to the LDL lipid surface. In order to understand the apoB-lipid interactions, we have synthesized several apoB peptide sequences that have a 5-amino-acid repeating motif, a unique structural feature of the proline-rich regions.
The binding of the apoB-derived peptides to small unilamellar lipid vesicles (SUVs) was primarily monitored by means of fluorescence measurements. A simple surface partition equilibrium was used to characterize the binding isotherms, and the resultant partition constants (K$\sb{\rm p}$) were compared with those of a model cationic amphiphilic $\alpha$-helical peptide and a model amphiphilic $\beta$-sheet peptide.
In an initial series of lipid binding experiments that measured the weak Phe fluorescence of the apoB peptides, a peptide length requirement of 40 residues was demonstrated for binding to electrically neutral phosphatidylcholine (PC) vesicles, and only weak additional electrostatic attractions to cationic 1,2-dioleoyl-3-trimethylammonium-propane (TAP) vesicles were observed. After accounting for the electrostatic attraction between the oppositely charged peptides and lipid vesicles using Gouy-Chapman theory, the intrinsic partition constant, K$\sb{\rm p}$, for the 40-residue apoB peptide was found to be 1.0-2.6 $\times$ 10$\sp3$ M$\sp{-1}.$ Similar K$\sb{\rm p}$ values of 2.8-4.0 $\times$ 10$\sp3$ M$\sp{-1}$ were determined for the 22-residue cationic model amphiphilic $\alpha$-helical peptide for binding to the electrically neutral PC vesicles. However, the helical peptide showed an additional stronger electrostatic attraction to anionic phosphatidylglycerol (PG) vesicles. The cationic 20-residue $\beta$-sheet peptide showed an even stronger electrostatic attraction to anionic PG vesicles, but it did not bind to electrically neutral PC vesicles, and calculated intrinsic partition constants, K$\sb{\rm p}$, for the anionic vesicles were small.
In a second series of lipid binding studies, a fluorescence probe (NBD) was incorporated into the apoB peptide sequences. These studies also demonstrated the dependence of the peptide affinities for neutral lipid vesicles on peptide length. Addition of cholesterol to the zwitterionic vesicles resulted in 2 to 5-fold weaker $\beta$-sheet peptide binding, also in contrast with the behavior of the amphiphilic $\alpha$-helical peptides.
Overall, these studies demonstrated for the first time that the proline-rich apoB structural motif of repeating 5-amino-acid homologous sequences can bind to neutral lipid vesicles. This supports the original proposal of a phospholipid binding function for these $\beta$-sheet structures.
