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 apolipo protein of 17 kDa that contains no Trp, one Tyr, and eight Phe. The ef fect of pH on the kinetics of association of apoLp-III with dimyristoy lphosphatidylcholine was studied. The pH dependence of the kinetics sh owed three distinct regions. Above pH 7, the reaction rate is slow and slightly affected by pH. A similar to 40-fold increase in the rate co nstant is observed when the pH is decreased from 8 to 4, and a decreas e in rate is observed below pH 4. Far-UV CD spectroscopy indicated tha t the secondary structure of the protein is not affected when decreasi ng the pH from 8 to 4.5, The pH dependence of the Tyr fluorescence sho wed three pH regions that resemble the regions observed in the kinetic s. Comparison of the far-UV CD and fluorescence studies indicated the formation of a folding intermediate between pHs 4 and 7. This intermed iate was also characterized by near-UV CD and fluorescence quenching. Fluorescence quenching studies with I- and Csf indicated a very low ex posure of the Tyr residue in both native and intermediate conformation s. The pH dependence of the near-UV CP spectra indicated that the nati ve --> 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 similar t o 4 Phe residues. This report shows, for the first time, the correlati on between the increase in lipid binding activity of an exchangeable a polipoprotein and the formation of a compact but hydrated conformation near physiological conditions. These results suggest a direct correla tion between the lipid binding activity and the internal hydration of the apolipoprotein. The similarity between the insect exchangeable apo lipoprotein 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 Atkinso n, D. (1996) Proc. Natl. Acad Sci. U.S.A. 93, 2991-2995] suggest that this highly hydrated and compact state may play an important physiolog ical role as the most active lipid binding state of the apolipoprotein s in general.