Fluorescence Quenching Studies of Apolipoprotein A-I in Solution and in Lipid—Protein Complexes: Protein Dynamics

William W. Mantulin, Henry J. Pownall, David M. Jameson

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

Fluorescence lifetime and intensity quenching studies of human plasma apolipoprotein A-I (apo A-I) in aqueous solution and in recombinant lipoprotein complexes with dimyristoylphosphatidylcholine (DMPC) indicate differences in conformational dynamics. In aqueous solution, the bimolecular quenching constants (k*) for lipid-free apo A-I fluorescence quenching by oxygen and acrylamide are 2.4 × 109 and 0.38 × 109 M-1 s-1, respectively. These values are independent of the oligomeric form of the protein. There is no correlation between the relatively small k* for apo A-I, which reflects rapid, low-amplitude protein fluctuations, and the labile conformational changes of apo A-I folding reactions, like denaturation, which occur on a slower time scale. In recombinant DMPC/apo A-I complexes (100:1 molar ratio) the protein increases in amphiphilic α-helical structure as it blankets the lipid matrix. The apparent k* for oxygen quenching of apo A-I fluorescence in the complex is large and increases in a temperature-dependent manner. We have introduced a two-compartment model, which discriminates the source of quencher molecules as aqueous or lipid, to describe oxygen quenching of DMPC/apo A-I fluorescence. The magnitude and temperature dependence of the apparent k* predominantly reflect the partitioning of oxygen between the two phases rather than being a probe of the lipid physical state. Calculations of the helical hydrophobic moment in apo A-I indicate that tryptophan residues 8 and 72 occur at the lipid-protein interface of amphiphilic α-helices, whereas the other two tryptophan residues (50, 108) lie on the nonpolar faces of amphiphilic helices. This placement of fluorescent residues, relative to the lipid-protein interface, suggests a temperature-dependent solvation barrier for oxygen quencher molecules originating in the lipid phase. The diminution of the temperature dependence of the oxygen quenching efficiency of DMPC/apo A-I fluorescence by the addition of sucrose, which perturbs the water-phospholipid-protein interfacial region, probably reflects changes in the solvation barrier. Quenching of DMPC/apo A-I intrinsic fluorescence by acrylamide is similar to that observed for lipid-free apo A-I. Analysis of this data does not require a two-compartment model. The acrylamide quenching results indicate that the increase in α-helical structure, experienced by apo A-I in lipid association, does not significantly alter the access of uncharged quenchers to fluorescent residues. Our studies demonstrate that the hydrated lipid-protein interface of lipoproteins is the major factor in regulating protein dynamics.

Original languageEnglish (US)
Pages (from-to)8034-8042
Number of pages9
JournalBiochemistry
Volume25
Issue number24
DOIs
StatePublished - Dec 1986

ASJC Scopus subject areas

  • Biochemistry

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