Amphipathic alpha-helix bundle organization of lipid-free chicken apolipoprotein A-I

Apolipoprotein A-I (apoA-I), the major protein component of plasma high-den sity lipoprotein (HDL), exists in alternate lipid-free and lipid-bound stat es. Among various species, chicken apoA-I possesses unique structural prope rties: it is a monomer in the lipid-free state and it is virtually the sole protein component of HDL. Near-UV circular dichroism (CD) spectroscopic st udies provide evidence that chicken apoA-I undergoes a major conformational change upon binding to lipid, while far-UV CD data indicate its overall al pha-helix content is maintained during this transition. The fluorescence em ission wavelength maximum (excitation 295 nm) of the tryptophans in apoA-I (W74 and W107) displayed a marked blue shift in both the lipid-free (331 nm ) and HDL-bound (329 nm) states, compared to free tryptophan in solution. T he effect of aqueous quenchers on tryptophan fluorescence was determined in lipid-free, dimyristoylphosphatidylcholine (DMPC)- and HDL-bound states. T he most effective quencher in the lipid-free and HDL-bound states was acryl amide, giving rise to K-sv values of 1.6 +/- 0.1 and 1.2 +/- 0.1 M-1, respe ctively. Together, these data suggest that a hydrophobic environment around the two tryptophan residues (W74 and W107) is maintained in alternate conf ormations of the protein. To further probe the molecular organization of li pid-free apoA-I, its effect on the fluorescence properties of 8-anilino-1-n aphthalenesulfonic acid (ANS) was determined. Human and chicken apoA-I indu ced a similar increase in ANS fluorescence quantum yield, in keeping with t he hypothesis that these proteins adopt a similar global fold in the absenc e of lipid. When considered with near- and far-UV CD experiments, the data support a model in which lipid-free chicken apoA-I is organized as an amphi pathic ct-helix bundle. In other studies, lipid-soluble quenchers, 5-, 7-, 10-, and 12-DOXYL stearic acid (DSA), were employed to investigate the dept h of penetration of apoA-I into the surface monolayer of spherical HDL part icles. 5-DSA was the most effective quencher, suggesting that apoA-I trypto phan residues localize near the surface monolayer, providing a structural r ationale for the reversibility of apoA-I-lipoprotein particle interactions.