STRUCTURAL ORGANIZATION OF LIPID PHASE AND PROTEIN-LIPID INTERFACE INAPOLIPOPROTEIN-PHOSPHOLIPID RECOMBINANTS - INFLUENCE OF CHOLESTEROL

The complexes of individual human plasma apolipoproteins (apo) A-I, E and A-II with dipalmitoylphosphatidylcholine (DPPC) in the absence or in the presence of cholesterol (Chol) were prepared with initial DPPC/ Chol/protein weight ratio as 3:0.15:1. ApoA-I/DPPC/Chol complexes with different protein content (initial DPPC/apoA-I weight ratios were cha nged from 10.5:1 to 2.6:1) but with a fixed initial DPPC/Chol weight r atio of 20:1 were also prepared. The complexes were isolated by gel-fi ltration and characterized by size and composition. ApoA-I- and apoA-I I-complexes had the same size (80-84 Angstrom) and the complexes becam e more heterogeneous upon Chol inclusion; apoE-complexes were larger ( 97-100 Angstrom) and more homogeneous and Chol addition had no effect on their hydrodynamic properties. Chol seems to be excluded partially in the following manner for isolated complexes with different apo's: A -II > E > A-I. The possible existence of two lipid regions in the comp lexes differing in lipid dynamics - the lipid shell in the vicinity of apolipoprotein (boundary lipid) opposite to the remaining part of the lipid bilayer - has been studied by absorbance and fluorescence spect roscopy with cis-parinaric acid (cis-PA) and trans-parinaric acid (tra ns-PA) embedded into the complexes. Their application is based on a st rong preference of ri-ans-PA for solid lipid while cis-PA distributes more equally between co-existing fluid and solid lipid regions (Sklar et al. (1979) Biochemistry 18, 1707-1716). (1) For apoA-I-complexes, t he partition of cis-PA between water and lipid phase at temperatures b elow and above the transition temperature of DPPC (T-t) was insensitiv e to Chol and temperature, while partition of trans-PA into the lipid phase of Chol-containing complex was increased at high temperature and decreased at low temperature. These results seem to be related to tra ns-PA redistribution between Chol-rich and protein-rich lipid domains, the latter being more disordered at T < T-t and more immobilized at T > T-t compared to the bulk bilayer; cis-PA localizes preferentially i n boundary lipid. This hypothesis was directly confirmed by measuremen ts of energy transfer between apoA-I tryptophanyls and probe molecules . (2) The relative response of trans-PA fluorescence intensity to temp erature-induced phase transition of DPPC in apoA-I/DPPC/Chol complexes was decreased as a function of apolipoprotein content in a non-monoto nic fashion with a transition midpoint at a mel ratio DPPC/A-I of 250: 1, probably indicating two different modes of apolipoprotein/DPPC inte raction in different sized complexes. (3) The comparative study of lip id dynamics in apoA-I-, apoE- and apoA-II-containing complexes with te mperature response to phospholipid phase transition with fluorescence parameters such as intensity and anisotropy of cis-PA and trans-PA rev ealed the presence of boundary lipid in all three complexes without Ch ol. In contrast to apoA-I-containing complexes, in apoA-II/DPPC/Chol c omplexes, trans-PA seems to move preferentially into boundary lipid an d cia-PA to distribute between two different regions probably as a res ult of more ordering action induced by apoA-II compared to apoA-I on t he nearest phospholipid molecules in Chol-containing complexes; the ap oE action on trans-PA and cia-PA distribution could be intermediate. B ased on these results, the degree of Chol exclusion from the boundary lipid region for complexes with different apo's increasing in the orde r A-II > E > A-I can be suggested. Different Chol distributions betwee n two lipid regions in the complexes seems not to be a function of com plex size, but rather is an inherent property of the particular apolip oprotein molecule.