Amphipathic alpha-helix bundle organization of lipid-free chicken apolipoprotein A-I.
Apolipoprotein A-I (apoA-I), the major protein component of plasma high-density lipoprotein (HDL), exists in alternate lipid-free and lipid-bound states. Among various species, chicken apoA-I possesses unique structural properties: 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 studies provide evidence that chicken apoA-I undergoes a major conformational change upon binding to lipid, while far-UV CD data indicate its overall alpha-helix content is maintained during this transition. The fluorescence emission 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. The effect of aqueous quenchers on tryptophan fluorescence was determined in lipid-free, dimyristoylphosphatidylcholine (DMPC)- and HDL-bound states. The most effective quencher in the lipid-free and HDL-bound states was acrylamide, giving rise to Ksv values of 1.6 +/- 0.1 and 1.2 +/- 0.1 M-1, respectively. Together, these data suggest that a hydrophobic environment around the two tryptophan residues (W74 and W107) is maintained in alternate conformations of the protein. To further probe the molecular organization of lipid-free apoA-I, its effect on the fluorescence properties of 8-anilino-1-naphthalenesulfonic acid (ANS) was determined. Human and chicken apoA-I induced a similar increase in ANS fluorescence quantum yield, in keeping with the hypothesis that these proteins adopt a similar global fold in the absence 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 amphipathic alpha-helix bundle. In other studies, lipid-soluble quenchers, 5-, 7-, 10-, and 12-DOXYL stearic acid (DSA), were employed to investigate the depth of penetration of apoA-I into the surface monolayer of spherical HDL particles. 5-DSA was the most effective quencher, suggesting that apoA-I tryptophan residues localize near the surface monolayer, providing a structural rationale for the reversibility of apoA-I-lipoprotein particle interactions.
Kiss
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Lipid and Lipoprotein Research Group, Protein Engineering Network of Centres of Excellence (PENCE), University of Alberta, Edmonton, Canada.
Kay
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Ryan
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D020106:Acrylamide / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry*; D002586:Cesium / Q000737:chemistry; D002645:Chickens; D002712:Chlorides / Q000737:chemistry; D002942:Circular Dichroism; D005227:Fatty Acids / Q000737:chemistry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D011193:Potassium Iodide / Q000737:chemistry; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D014270:Trifluoroethanol / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D016632:Apolipoprotein A-I; D002712:Chlorides; D005227:Fatty Acids; D005456:Fluorescent Dyes; D008055:Lipids; D013113:Spin Labels; D011193:Potassium Iodide; D002586:Cesium; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; D014270:Trifluoroethanol; D014364:Tryptophan; C028019:cesium chloride