Disruption of the C-terminal helix by single amino acid deletion is directly responsible for impaired cholesterol efflux ability of apolipoprotein A-I Nichinan.

Apolipoprotein A-I (apoA-I) Nichinan, a naturally occurring variant with DeltaE235 in the C terminus, is associated with low plasma HDL levels. Here, we investigated the tertiary structure, lipid-binding properties, and ability to induce cellular cholesterol efflux of apoA-I Nichinan and its C-terminal peptide. Thermal and chemical denaturation experiments demonstrated that the DeltaE235 mutation decreased the protein stability compared with wild type (WT).

Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles.

The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and -unbound states is an integral part of HDL metabolism. We used the surface plasmon resonance (SPR) technique to monitor in real time the reversible binding of apoA-I to HDL. Biotinylated human HDL(2) and HDL(3) were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were flowed across the surface.

Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I.

Carriers of the apolipoprotein A-I(Milano) (apoA-I(M)) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I(M) mutation: R173S apoA-I, similar to apoA-I(M) but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation.

Conformational flexibility of the N-terminal domain of apolipoprotein a-I bound to spherical lipid particles.

Lipid binding of human apolipoprotein A-I (apoA-I) occurs initially through the C-terminal alpha-helices followed by conformational reorganization of the N-terminal helix bundle. This led us to hypothesize that apoA-I has multiple lipid-bound conformations, in which the N-terminal helix bundle adopts either open or closed conformations anchored by the C-terminal domain. To investigate such possible conformations of apoA-I at the surface of a spherical lipid particle, site-specific labeling of the N- and C-terminal helices in apoA-I by N-(1-pyrene)maleimide was employed after substitution of a Cys residue for Val-53 or Phe-229.