Investigation of the lipid domains and apolipoprotein orientation in reconstituted high density lipoproteins by fluorescence and IR methods.
The reconstituted high density lipoproteins (rHDL) that were described in the preceding paper (Hefele Wald, J., Krul, E. S., and Jonas, A. (1990) J. Biol. Chem. 265, 20037-20043) are used in this study to analyze the organization, conformation, and dynamics of the lipid phase, as well as the relative orientation of the apolipoprotein alpha-helices and the lipid hydrocarbon chains. Two fluorescence polarization probes and a fluorescence polarity probe were used to detect the lipid phase transition behavior of the various particles, and to estimate the lipid order, mobility, and environment polarity in their gel and liquid-crystalline states. Infrared attenuated total reflection spectroscopy was used to estimate the content of secondary structure of the apolipoprotein, and the orientation of its alpha-helices with respect to the lipid hydrocarbon chains. In addition, the infrared spectra were analyzed in terms of the conformation and organization of different regions of the lipid molecules in the rHDL particles. The results indicate that the overall organization and conformation of lipid molecules in a lipid bilayer is preserved in the rHDL particles, but that progressive increases in apolipoprotein content straighten the hydrocarbon chains and decrease their packing order in the gel state, and decrease their mobility in the liquid-crystalline state. The presence of apolipoprotein also affects the conformation of the lipids at the level of the ester bonds and the head group of the phospholipid. In all three particle classes the content and distribution of secondary structures of the apolipoprotein were similar, and the alpha-helical segments were parallel to the lipid hydrocarbon chains.
Wald
J H
JH
Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois 61801.
Coormaghtigh
E
E
De Meutter
J
J
Ruysschaert
J M
JM
Jonas
A
A
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D015081:2-Naphthylamine / Q000031:analogs & derivatives; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000737:chemistry*; D002942:Circular Dichroism; D004161:Diphenylhexatriene / Q000031:analogs & derivatives; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006801:Humans; D008051:Lipid Bilayers / Q000032:analysis; D008055:Lipids / Q000737:chemistry*; D008077:Lipoproteins, LDL / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008055:Lipids; D008077:Lipoproteins, LDL; D004161:Diphenylhexatriene; D015060:1,2-Dipalmitoylphosphatidylcholine; C038517:prodan; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D015081:2-Naphthylamine