Changes in helical content or net charge of apolipoprotein C-I alter its affinity for lipid/water interfaces

Abstract

Amphipathic α-helices mediate binding of exchangeable apolipoproteins to lipoproteins. To probe the role of α-helical structure in protein-lipid interactions, we used oil-drop tensiometry to characterize the interfacial behavior of apolipoprotein C-I (apoC-I) variants at triolein/water (TO/W) and 1-palmitoyl-2-oleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. ApoC-I, the smallest apolipoprotein, has two amphipathic α-helices. Mutants had single Pro or Ala substitutions that resulted in large differences in helical content in solution and on phospholipids. The ability of apoC-I to bind TO/W and POPC/TO/W interfaces correlated strongly with α-helical propensity. On binding these interfaces, peptides with higher helical propensity increased surface pressure to a greater extent. Likewise, peptide exclusion pressure at POPC/TO/W interfaces increased with greater helical propensity. ApoC-I retention on TO/W and POPC/TO/W interfaces correlated strongly with phospholipid-bound helical content. On compression of these interfaces, peptides with higher helical content were ejected at higher pressures. Substitution of Arg for Pro in the N-terminal α-helix altered net charge and reduced apoC-I affinity for POPC/TO/W interfaces. Our results suggest that peptide-lipid interactions drive α-helix binding to and retention on lipoproteins. Point mutations in small apolipoproteins could significantly change α-helical propensity or charge, thereby disrupting protein-lipid interactions and preventing the proteins from regulating lipoprotein catabolism at high surface pressures.

Keywords: drop tensiometry; protein-lipid interaction; surface chemistry.

Fig. 1.

Helical wheel representations of the two predicted apoC-I amphipathic α-helices. Helices are modeled as 11/3 helices (13). By convention, apolar residues are colored in yellow, polar in gray, basic in blue, acidic in red, and glycine in pink. The hydrophobic face of each helix is indicated by dotted lines. Amino acids substituted for Ala or Pro are shown by red circles.

Fig. 2.

Peptide-induced remodeling of various lipid surfaces. (A) Interfacial tension (γ) versus time curves for apoC-I variants at TO/W interfaces. TO drops (16 μl) were formed in 6.0 ml of phosphate buffer, and peptides were added to the aqueous phase at 2.5 × 10⁻⁷ M. Changes in γ as peptides adsorbed to TO drops were approximated by sigmoidal functions, where transition width t_1/2 is the midpoint corresponding to 50% decrease in γ due to peptide adsorption as depicted for G15A. (B) Interfacial tension versus time curves for apoC-I variants at POPC/TO/W interfaces. POPC was adsorbed to 16 μl TO drops and γ reached ∼24 to 25 mN/m after buffer exchange. Peptides were added to the aqueous phase at 2.5 × 10⁻⁷ M at arbitrary time 0 sec. The changes in γ as peptides adsorbed to POPC/TO drops were approximated by sigmoidal functions.

Fig. 3.

Examples of desorption and readsorption curves of G15A at TO/W (A) and POPC/TO/W (B) interfaces. (A) G15A was added to the aqueous phase at 1.3 × 10⁻⁷ M. After G15A adsorption to a 16 μl TO drop lowered γ to γ_eq = 13.5 mN/m, the drop was compressed and subsequently reexpanded in increments of ±2 μl, ±4 μl, ±5 μl, ±6 μl, and ±8 μl. G15A was removed from the aqueous phase by a 150 ml buffer exchange (shown by the bar). The drop was compressed and reexpanded in increments of ±4 μl, ±6 μl, and ±8 μl. (B) A 16 μl TO drop was formed and POPC adsorption lowered γ to 25.2 mN/m. POPC was removed from the aqueous phase by a 250 ml buffer exchange (shown by the first bar). G15A was added to the aqueous phase at 1.3 × 10⁻⁷ M, and adsorption lowered γ to γ_eq = 9.5 mN/m. The TO drop was compressed and reexpanded in increments of ±2 μl, ±4 μl (×2), ±5 μl, and ±6 μl. G15A was removed from solution by a 150 ml buffer exchange (shown by the second bar). The drop was compressed and reexpanded in increments of ±2 μl, ±6 μl, ±8 μl, and ±10 μl.

Fig. 4.

Π_MAX of apoC-I variants at TO/W (A) and POPC/TO/W (B) interfaces. Each interface was rapidly compressed to Π_o. The TO drop was held at that compressed volume for several minutes, and the resulting Δγ was plotted against Π_o. Linear regression of the data at each interface gave x-intercepts at Δγ = 0 mN/m, marked by a box. These linear regressions were significant (0.88 < R < 0.99, P < 0.0001). X-intercepts represent Π_MAX, the pressure at which peptides show no net desorption from the given interface. The data points for each peptide are from several compression and expansion experiments with aqueous peptide concentrations of 1.3–5.0 × 10⁻⁷ M.

Fig. 5.

Pressure-area (Π-A) isotherms for apoC-I variants G15A (a), WT (b), G15P (c), R23P (d), and M38P (e) adsorbed to a POPC/TO/W interface of Г_POPC = 36.5 ± 0.5%. Peptides, added at 1.3 × 10⁻⁷ M in the aqueous phase, adsorbed to POPC/TO/W interfaces. After buffer exchange, the interface was slowly expanded and sequentially compressed. Shown here are compression isotherms, with an arrow marking the direction of compression. Asterisks mark envelope points, where an abrupt change in slope shows the beginning of ejection of the given peptide from the surface. No peptide (f) is the Π-A compression isotherm for the peptide-free POPC/TO/W interface.

Fig. 6.

Π_ENV dependence on Г_POPC for the apoC-I variants. Peptides, added at 1.3 × 10⁻⁷ M in the aqueous phase, adsorbed to a TO/W interface or POPC/TO/W interfaces at varied Г_POPC values. After buffer exchange, the interface was slowly expanded and compressed. Envelope pressures (Π_ENV) were found and plotted against Г_POPC. Π_ENV values at a TO/W interface (Г_POPC = 0%) are marked by a box. Each Π_ENV represents the average of n = 2–4 experiments. As a result, y-error bars are the standard deviation in Π_ENV, and x-error bars are the uncertainty in Γ_POPC extrapolated from the standard deviation in Π_i. Linear regressions applied to the data were significant (0.96 < R < 0.99, P < 0.003), but Π_ENV at a TO/W interface was not included in the linear regression (dashed line) for R23P data.

Fig. 7.

Π_EX of apoC-I variants at POPC/TO/W interfaces. For a given peptide, adsorption to a TO/W interface of Π_i = 0 mN/m, Г_POPC = 0%, or POPC/TO/W interfaces of various Π_i or Г_POPC values increased ΔΠ to a different extent. For each peptide, ΔΠ values were plotted against corresponding Π_i values. Linear regression of the data gave x-intercepts at ΔΠ = 0 mN/m, marked by a box. These linear regressions were significant (−0.99 < R < −0.96, P < 0.0001). X-intercepts represent Π_EX, the exclusion pressure at which a given peptide cannot bind POPC/TO/W interfaces.

Fig. 8.

Schematic model of apoC-I variants binding to and retention on POPC/TO/W interfaces. The NMR structure of WT apoC-I (Protein Data Bank entry 1IOJ) (18) is colored according to secondary structure, and to represent less-folded states, is modified to exhibit reduced helical content in its N-terminus. POPC has red polar groups and yellow hydrocarbon tails. TO is represented by a transparent yellow sphere. In solution, WT and G15A apoC-I have a fluctuating helix-loop-helix conformation (A), but R23P, G15P, and M38P apoC-I are largely unfolded (B). (A) On binding to a POPC/TO/W interface (left), WT and G15A increase in helical content, form extensive peptide-lipid interactions, and increase surface pressure. On small interfacial compressions (middle), bound apoC-I is compressed and its N-terminal α-helix may desorb from the surface (47). When the interface is compressed above the peptide's retention pressure (right), peptide desorbs from the surface. (B) R23P, G15P, and M38P, bound to a POPC/TO/W interface (left), have less helical content than WT, resulting in less extensive peptide-lipid interactions and smaller pressure changes. On interfacial compressions (middle), R23P, G15P, and M38P have lower retention pressures than WT and are ejected from the interface at lower pressures. As the surface pressure of POPC/TO/W interfaces increases (right), these peptides are excluded from the interface at lower pressures than WT.