Fluorescence analysis of the lipid binding-induced conformational change of apolipoprotein E4

Abstract

Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipid-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL(3) suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL(3).

Fig. 1

(A) Acrylodan fluorescence emission spectra of apoE4 S94C-acrylodan at different concentrations of GdnHCl (from 0 to 4.6 M). (B) The change in acrylodan GP for apoE4 S94C-acrylodan (Δ), W264C-acrylodan (○), and S290C-acrylodan (●) as a function of GdnHCl concentration. Protein concentration was 25 µg/ml.

Fig. 2

Pyrene fluorescence emission spectra of apoE4 S94C-pyrene (A) and S290C-pyrene (B) at different concentrations of proteins (5–100 µg/ml). (C) The pyrene excimer (at 470 nm)/monomer (at 375 nm) intensity ratio for apoE4 S94C-pyrene (Δ), W264C-pyrene (○), and S290C-pyrene (●) as a function of apoE4 concentration. The excitation wavelength was 342 nm.

Fig. 3

Pyrene fluorescence emission spectra of apoE4 S290C-pyrene (25 µg/ml) at different concentrations of GdnHCl (from 0 to 2 M). The inset shows the change in the excimer/monomer ratio as a function of GdnHCl concentration. The excitation wavelength was 342 nm.

Fig. 4

(A) Pyrene fluorescence emission spectra of apoE S290C-pyrene in the lipid-free (dashed line) or bound to egg PC SUV (solid line). The arrow draws attention to the excimer peak at around 470 nm. Protein and PC concentrations were 25 µg/ml and 1.5 mg/ml, respectively. (B) Increases in fluorescence intensity of apoE4 S94C-pyrene (Δ), W264C-pyrene (○), and S290C-pyrene (▲) upon binding to egg PC SUV as a function of the weight ratio of PC to apoE4. Protein concentration was 25 µg/ml. The inset shows the linearized plots according to Hanes-Woolf equation (see Experimental Procedures). (C) Time courses of increases in fluorescence intensity upon binding to egg PC SUV for apoE4 S94C-pyrene (trace a), W264C-pyrene (trace b), and S290C-pyrene (trace c). SUV was added to apoE4 variants at final concentrations of 10 µg/ml protein and 0.4 mg/ml PC. Pyrene fluorescence was monitored at 385 nm with an excitation of 342 nm.

Fig. 5

(A) FRET between Trp residues and acrylodan in apoE4 S94C-acrylodan. Fluorescence emission spectra excited at 290 nm were recorded in the lipid-free (solid line) or SUV-bound (dashed line) states. Protein and PC concentrations were 25 µg/ml and 1.0 mg/ml, respectively. (B) Increases in fluorescence intensity ratio of Trp residues at 340 nm to acrylodan at 480 nm as a function of the weight ratio of PC to apoE4. Protein concentration was 25 µg/ml. The inset shows the linearized plots according to Hanes-Woolf equation. (C) Comparison of time courses of increase in pyrene fluorescence intensity and fluorescence intensity ratio of Trp to acrylodan for apoE4 S94C-acrylodan upon binding to egg PC SUV.

Fig. 6

Time courses of increases in fluorescence intensity upon binding to VLDL (A) and HDL₃ (B) for apoE4 S94C-pyrene (trace a) and W264C-pyrene (trace b). VLDL or HDL₃ was added to apoE4 variants at final concentrations of 10 µg/ml apoE4 and 0.1–0.4 mg/ml PL. Pyrene fluorescence was monitored at 385 nm with an excitation of 342 nm. (C) Increases in fluorescence intensity of apoE4 S94C-pyrene (Δ), W264C-pyrene (○), and S290C-pyrene (●) upon binding to HDL₃ as a function of the weight ratio of PL to apoE4. Protein concentration was 25 µg/ml.