Pyrene Excimer Fluorescence:  A Spatially Sensitive Probe To Monitor Lipid-Induced Helical Rearrangement of Apolipophorin III

Abstract

Manduca sexta apolipophorin III (apoLp-III), an 18-kDa, monomeric, insect hemolymph apolipoprotein, is comprised of five amphipathic alpha-helices arranged as a globular bundle in the lipid-free state. Upon lipid binding, it is postulated that the bundle opens, exposing a continuous hydrophobic surface which becomes available for lipid interaction. To investigate lipid binding-induced helical rearrangements, we exploited the unique fluorescence characteristics of N-(1-pyrene)maleimide. Pyrene is a spatially sensitive extrinsic fluorescent probe, which forms excited-state dimers (excimers) upon close encounter with another pyrene molecule. Cysteine residues were introduced into apoLp-III (which otherwise lacks cysteine) at Asn 40 (helix 2) and/or Leu 90 (helix 3), creating two single-cysteine mutants (N40C-apoLp-III and L90C-apoLp-III) and N40C/L90C-apoLp-III, a double-cysteine mutant, which were labeled with pyrene maleimide. Pyrene-labeled N40C/L90C-apoLp-III, but not the pyrene-labeled single-cysteine mutants, exhibited strong excimer fluorescence in the lipid-free, monomeric state. Guanidine hydrochloride titration and temperature studies revealed a loss in excimer fluorescence, accompanied by a loss in the molar ellipticity of the protein. When apoLp-III interacts with phospholipid vesicles to form disklike complexes, a significant loss in excimer fluorescence was noted, indicating that the helices bearing the pyrene moieties diverge from each other. Pyrene excimer fluorescence was further employed to examine the relative orientation of lipid-bound apoLp-III molecules. Pyrene-labeled N40C- or L90C-apoLp-III displayed no excimer fluorescence in the disk complexes, while complexes prepared with an equal mixture of both single-labeled mutants did emit excimer fluorescence, indicating apoLp-III adopts a preferred nonrandom orientation around the perimeter of the bilayer disk. These studies establish pyrene excimer fluorescence as a useful spectroscopic tool to address intra- and intermolecular interactions of exchangeable apolipoproteins upon binding to lipid.

Examination of Lipid-bound Conformation of Apolipoprotein E4 by Pyrene Excimer Fluorescence

“…However, when there is a second bound pyrene within 10 Å in spatial proximity, it displays an additional broad and red-shifted fluorescence emission peak (ϳ460 nm), attributed to formation of an excited state dimer or 'excimer' (37). We exploit this unique spectral feature of pyrene to assess lipid-triggered repositioning of two specific sites, one in NT and the other in CT domain of apoE4, in an approach successfully employed to evaluate spatial proximity in other proteins (37)(38)(39)(40) including apolipoproteins such as apolipophorin III (31,41). Furthermore, when two pyrene molecules giving rise to excimer fluorescence move away from each other, the excimer fluorescence emission intensity is decreased or lost entirely.…”

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

“…When attached to a protein thiol, the dye produces two distinct emission peaks at 375 and 395 nm. When two pyrene-thiols interact within 6 -10 Å distance, they form excited state dimers (excimers) that emit at longer wavelengths than the lone excited fluorophore (24,25). The transporter labeled with the pyrene probe at the cysteine positions described above retained ϳ80% ATPase activity and 70 -80% maltose transport activity as compared with the wild type complex ( Table 2).…”

Glycolipid transfer protein: Clear structure and activity, but enigmatic function

“…The glycolipid transfer activity of GLTP from donor to acceptor vesicles in vitro is routinely monitored using a fluorescence based resonance energy transfer assay (Abe et al, 1984;Mattjus et al, 1999;Sahoo et al, 2000). Self-quenching due to excimer formation of pyrene-labeled lipids is also used to study lipid transport (Sahoo et al, 2000;Somerharju, 2002).…”

“…Self-quenching due to excimer formation of pyrene-labeled lipids is also used to study lipid transport (Sahoo et al, 2000;Somerharju, 2002). We applied this technique to study single turnover transfer of partially pyrene-labeled glycolipid (GlcCerpyr and GlcCer mixed in 1:5 molar ratio) from donor vesicles to GLTP only.…”