The lipid binding activity of the exchangeable apolipoprotein apolipophorin-III correlates with the formation of a partially folded conformation.
Manduca sexta apolipophorin-III, apoLp-III, is an exchangeable apolipoprotein of 17 kDa that contains no Trp, one Tyr, and eight Phe. The effect of pH on the kinetics of association of apoLp-III with dimyristoylphosphatidylcholine was studied. The pH dependence of the kinetics showed three distinct regions. Above pH 7, the reaction rate is slow and slightly affected by pH. A approximately 40-fold increase in the rate constant is observed when the pH is decreased from 8 to 4, and a decrease in rate is observed below pH 4. Far-UV CD spectroscopy indicated that the secondary structure of the protein is not affected when decreasing the pH from 8 to 4.5. The pH dependence of the Tyr fluorescence showed three pH regions that resemble the regions observed in the kinetics. Comparison of the far-UV CD and fluorescence studies indicated the formation of a folding intermediate between pHs 4 and 7. This intermediate was also characterized by near-UV CD and fluorescence quenching. Fluorescence quenching studies with I- and Cs+ indicated a very low exposure of the Tyr residue in both native and intermediate conformations. The pH dependence of the near-UV CD spectra indicated that the native --> intermediate transition is accompanied by a loss in the packing constrains of the Tyr residue. UV absorption spectroscopy of the Phe and Tyr residues indicated that the native --> intermediate transition is also accompanied by the hydration of the Tyr residue and approximately 4 Phe residues. This report shows, for the first time, the correlation between the increase in lipid binding activity of an exchangeable apolipoprotein and the formation of a compact but hydrated conformation near physiological conditions. These results suggest a direct correlation between the lipid binding activity and the internal hydration of the apolipoprotein. The similarity between the insect exchangeable apolipoprotein and the human counterparts, apoA-I, apoA-II, etc., and the recent demonstration of the presence of a molten globular like-state of human apoA-I near physiological conditions [Gursky, O., and Atkinson, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 2991-2995] suggest that this highly hydrated and compact state may play an important physiological role as the most active lipid binding state of the apolipoproteins in general.
Department of Biochemistry, University of Arizona, Tucson 85721, USA. jlsoulag@u.arizona.edu
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D000818:Animals; D001053:Apolipoproteins / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D018402:Manduca; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry
D001053:Apolipoproteins; D002352:Carrier Proteins; C042470:apolipophorin III; D014443:Tyrosine; D004134:Dimyristoylphosphatidylcholine