MICROENVIRONMENTS OF BASIC-AMINO-ACIDS IN AMPHIPATHIC ALPHA-HELICES BOUND TO PHOSPHOLIPID - C-13 NMR-STUDIES USING SELECTIVELY LABELED PEPTIDES

The lipid-binding properties of serum apolipoproteins are mediated by class A amphipathic alpha-helices in which basic and acidic amino acid residues tend to be localized at the helix polar-nonpolar boundary and the center of the polar face, respectively. To better understand the role of the interfacial basic residues, the microenvironments and titration characteristics of lysine (Lys) residues have been examined using two 18-residue peptides. The parent 18A molecule (AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe) binds phospholipid relatively well because of its class A amphipathic helix, whereas 18R, which has the positions of basic and acidic residues reversed, binds to phospholipid relatively weakly, Lys residues were C-13-dimethylated, and NMR spectra were obtained of the peptides complexed with dimyristoylphosphatidylcholine (1/1 (w/w) DMPC/peptide). The four [C-13]-epsilon-dimethyl-Lys in 18A gave four resonances at chemical shifts (delta) of 42-43 ppm, whereas only a single resonance at delta 42.70 ppm was observed from the four Lys in 18R, Measurements of delta as a function of pH gave pK(a) values. The four Lys in 18R had a common pK(a) of 10.3, indicating that the Lys in the polar face of the amphipathic helix have the pK(a) expected for noninteracting, fully hydrated groups. In contrast, the four Lys in Ac-18A-NH2 had pK(a),'s in the range 9.4-11.0, indicating that they exist in various microenvironments at the polar-nonpolar boundary of the 18A helix. To examine individual Lys, 18A was synthesized with various Lys selectively labeled. Lys-4, -9, -13, and -15 in Ac-18A-NH2 associated with DMPC had pK(a) values of 11.0, 9.4, 9.4, and 10.3, respectively, The various Lys pK(a) values in 18A or Ac-18A-NH2 are determined primarily by interhelix interactions between antiparallel peptide molecules. In this configuration, the pK(a)'s of Lys-4 and Lys-15 are increased by the formation of attractive amino-aromatic and ion-pair interactions, respectively. Lys-9 and Lys-13, which are positioned on the opposite side of the amphipathic helix, experience a basic microenvironment that decreases their pK(a)'s. Novel insights into interactions between class A amphipathic helices in lipoprotein particles can be gained by the approach used in this study.