FLUORESCENCE STUDIES OF LIPID ASSOCIATION-INDUCED CONFORMATIONAL ADAPTATIONS OF AN EXCHANGEABLE AMPHIPATHIC APOLIPOPROTEIN

The conformational adaptability of Manduca sexta apolipophorin III (ap oLp-III) has been evaluated by monitoring the spectroscopic properties of its sole tyrosine residue, Tyr(145), present in the fifth helical segment of the protein. M. sexta apoLp-III adopts a globular five-heli x bundle structure in solution and has been postulated to undergo an o pening at putative hinge domains upon interaction with lipid surfaces. Previous results have shown that the intrinsic fluorescence of Tyr(14 5) iz highly quenched in the closed, water-soluble conformation but is dramatically enhanced upon lipid association. We have carried out a s pectroscopic characterization of Tyr(145) and its microenvironment, to enable its use as a structural probe of lipid-induced conformational changes of apoLp-III. The pK(a) of Tyr(145) in lipid-free apoLp-III wa s found to be 10.5, as determined from uv-spectrophotometry, indicatin g that, in the ground state, the tyrosyl phenolic group is not ionized under physiological conditions. Compared to free tyrosine in aqueous buffer (pH 7.0), a red shift (77 nm) in the lambda(max) of absorbance of Tyr(145) was observed, suggesting that an H-bonding interaction is responsible for the quenched state of tyrosine fluorescence. In an eff ort to explain the observed quenching phenomenon, the quantum yield an d lifetimes of Tyr(145) fluorescence emission were investigated as a f unction of pH and lipid binding. The quantum yield of Tyr(145) in lipi d-free apoLp-III was enhanced fivefold upon decreasing the pH, with a half-maximal point around pH 5.5. Time-resolved fluorescence decay ana lysis showed that Tyr(145) exhibits nonexponential emission decay with two components having lifetimes of 3.3 ns (76%) and 0.89 ns (24%) in the lipid-free state. The lifetime and amplitude of Tyr(145) remain es sentially unaltered upon lipid association or decreasing the pH. This is consistent with the hypothesis that, in the lipid-free helix bundle conformation, a quenching residue exists within H-bonding distance of the phenolic side chain of Tyr(145) which, at physiological pH, is re sponsible for the observed fluorescence quenching. Opening of the heli x bundle repositions this acceptor base, possibly a carboxylate or an imidazole side chain, making it unavailable for quenching. Using diffe rential polarized phase and modulation fluorometry, it was seen that t he segmental motion of Tyr(145) is also altered considerably upon lipi d interaction. These spectroscopic and motional properties of Tyr(145) distinguish this unique residue as a useful probe to monitor structur al flexibility of apoLp-III. (C) 1996 Academic Press, Inc.