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The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K(a)), the number of protein molecules bound per vesicle (n), enthalpy change (DeltaH degrees), entropy change (DeltaS degrees ), and the heat capacity change (DeltaC(p) degrees). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 degrees C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the alpha-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of POPC/FC at a molar ratio of 6/1 at (2.8-4) x 10(6) M(-1) and 2.4 apoA-I molecules bound per vesicle or 1.4 x 10(2) phospholipids per bound apoA-I. A plot of DeltaH degrees against temperature displayed a linear behavior, from which the DeltaC(p) degrees per mole of bound apoA-I was calculated to be -2.73 kcal/(mol x K). These results suggested that binding of apoA-I to POPC vesicles is characterized by nonclassical hydrophobic interactions, with alpha-helix formation as the main driving force for the binding to cholesterol-containing vesicles. In addition, comparison to literature data on peptides suggested a cooperativity of the helices in apoA-I in lipid interaction.

Two new Cys mutants of proapolipoprotein A-I, D9C and A232C, were created and expressed in Escherichia coli systems. Specific labeling with the thiol-reactive fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), was used to study the structural organization and dynamic properties of the extreme regions of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound states. Spectroscopic approaches, including circular dichroism and various fluorescence methods, were used to examine the properties of the mutant proteins and of their covalent adducts with the fluorescence probe. The mutations themselves had no effect on the structure and stability of apoA-I in the lipid-free state and in reconstituted HDL (rHDL) complexes. Furthermore, covalent modification with acrylodan did not alter the properties of the apoA-I variants in the lipid-bound state nor in the lipid-free A232C mutant, but it affected the structure and local stability of the lipid-free protein in the D9C mutant. Fluorescence results using the acrylodan probe confirmed a well-organized structure in the N-terminal region of apoA-I. Also, they suggested a three-dimensional structure in the C-terminal region, stabilized by protein-protein contacts. When Trp residues and acrylodan were used as donor-acceptor pairs for fluorescence resonance energy transfer (FRET), average distances could be measured. Both intensity and lifetime changes of the Trp emission indicated a protein folding in solution that brings the C-terminus of the protein near the Trp residues in the N-terminal half of the sequence. Also, the N- and C-terminal domains of apoA-I appeared to be near each other in rHDL having two apoA-I per particle.