Nanodiscs are phospholipid-protein complexes which are relevant to nascent high-density lipoprotein and are applicable as a drug carrier and a tool to immobilize membrane proteins. We evaluated the structure and dynamics of the nanoparticles consisting of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (apoA-I) with small-angle neutron scattering (SANS) and fluorescence methods and compared them with static/dynamic properties for large unilamellar vesicles. SANS revealed that the nanodisc includes a lipid bilayer with a thickness of 44 A and a radius of 37 A, in which each lipid occupies a smaller area than the reported molecular area of DMPC in vesicles. Fluorescence measurements suggested that DMPC possesses a lower entropy in nanodiscs than in vesicles, because apoA-I molecules, which surround the bilayer, force closer lipid packing, but allow water penetration to the acyl chain ends. Time-resolved SANS experiments revealed that nanodiscs represent a 20-fold higher lipid transfer via an entropically favorable process. The results put forward a conjunction of static/dynamic properties of nanodiscs, where the entropic constraints are responsible for the accelerated desorption of lipids.
Discoidal high-density lipoprotein (HDL) particles are known to be fractionalized into several discrete populations in plasma and to differ in behavior according to size; however, their structural differences and the factors regulating their size are less understood. In this study, we prepared several reconstituted HDLs (rHDLs) for structural evaluation by gel filtration chromatography and fluorometric analyses. With initial ratios of phospholipid (PL) to apolipoprotein A-I (apoA-I) between 25:1 and 100:1, unsaturated PLs constructed rHDLs with diameters of 9.5-9.6, 8.8-9.0, and 7.8-7.9 nm. Conversely, saturated PLs formed only the largest type of rHDLs (9.5-9.9 nm). While the largest rHDL comprised 23% cholesterol (Chol), the smallest rHDL contained only 13% Chol, which approximates liquid-ordered phase composition. As the size of rHDLs decreased, both the lateral pressure in the lipid bilayer, as determined from the excimer fluorescence of dipyrenylphosphatidylcholine, and the degree of hydration of the membrane surface, which was examined using the mean fluorescence lifetime of dansyl phosphatidylethanolamine, decreased well below the values obtained for large unilamellar vesicles. These results demonstrated that smaller rHDLs form a saddle surface, distinct from the planar bilayer produced by the largest forms.
The class A amphipathic α-helical peptide 18A is known to form discoidal phospholipid complexes (nanodiscs) similar to that formed by apolipoprotein A-I (apoA-I). To reveal the structural differences in nanodiscs formed with this protein and peptide, we prepared nanodiscs with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and applied fluorescence techniques to these nanoparticles. Fluorescence resonance energy transfer between 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) in nanodiscs revealed that lipid exchange with 18A nanodiscs is mediated by collisions between nanodiscs. The fluorescence lifetime of dansyl phosphatidylethanolamine and excimer fluorescence of 1,2-bis(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine showed that the degree of hydration of the membrane surface and lateral pressure of acyl chains in 18A nanodiscs are independent of the disc size, suggesting that 18A nanodiscs form planar lipid bilayers irrespective of their size, which differs from apoA-I nanodiscs, whose bilayer deforms to a saddle surface with decreasing size. These results suggest that the flexible structure of a chain of helices in apoA-I is crucial for the formation of saddle surfaces in nanodiscs.
The molecular mechanism by which nascent HDL forms via the interaction of apolipoprotein A-I (apoA-I) and transmembrane ABCA1 is poorly understood. Here, because ABCA1 has been reported to localize to acidic intracellular compartments, including the Golgi and endosome, we studied the interaction of apoA-I with model membranes under acidic conditions. Pure phosphatidylcholine liposomes were persistent against apoA-I at pH levels above 5.0, but were progressively transformed into reconstituted HDLs (rHDLs) by apoA-I at lower pH. Circular dichroism spectral measurements and 8-anilino-1-naphthalenesulfonic acid fluorescence measurements of lipid-free apoA-I ascribed this accelerated rHDL formation to the conformational change of the protein into a rather hydrophobic a-helical structure under acidic conditions. The addition of phosphatidylserine (PS) increased acidity at the bilayer surface and enabled the formation of discoidal rHDLs even at the pH of the endosome and slightly lower pH of the Golgi. These results suggest the following new scenario of nascent HDL formation: ABCA1, which colocalizes with apoA-I in acidic intracellular compartments, including the Golgi and endosome, increases acidity at the membrane surface on the luminal side by PS translocase activity and causes apoA-I to form nascent HDL.-Fukuda, M., M. Nakano, M. Miyazaki, M. Tanaka, H. Saito, S. Kobayashi, M. Ueno,andT.Handa.Conformational change of apolipoprotein A-I and HDL formation from model membranes under intracellular acidic conditions. J. Lipid Res. 2008Res. . 49: 2419Res. -2426
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