Structural and dynamic interfacial properties of the lipoprotein initiating domain of apolipoprotein B

Abstract

To better understand the earliest steps in the assembly of triglyceride (TG)-rich lipoproteins, we compared the biophysical and interfacial properties of two closely related apolipoprotein B (apoB) truncation mutants, one of which contains the complete lipoprotein initiating domain (apoB20.1; residues 1-912), and one of which, by virtue of a 50 amino acid C-terminal truncation, is incapable of forming nascent lipoproteins (apoB19; residues 1-862). Spectroscopic studies detected no major differences in secondary structure, and only minor differences in conformation and thermodynamic stability, between the two truncation mutants. Monolayer studies revealed that both apoB19 and apoB20.1 bound to and penetrated egg phosphatidylcholine (EPC) monolayers; however, the interfacial exclusion pressure of apoB20.1 was higher than apoB19 (25.1 mN/m vs. 22.8 mN/m). Oil drop tensiometry revealed that both proteins bound rapidly to the hydrophobic triolein/water interface, reducing interfacial tension by approximately 20 mN/m. However, when triolein drops were first coated with phospholipids (PL), apoB20.1 bound with faster kinetics than apoB19 and also displayed greater interfacial elasticity (26.9 +/- 0.8 mN/m vs. 22.9 +/- 0.8 mN/m). These data establish that the transition of apoB to assembly competence is accompanied by increases in surface activity and elasticity, but not by significant changes in global structure.

Fig. 1.

Amino acid sequence of the intervening peptide between apolipoprotein B (apoB)19 and apoB20.1. The bold black box indicates the location of a predicted α-helix. The two light-gray boxes indicate the location of predicted tilted fusogenic peptides.

Fig. 2.

CD spectra of apoB19 and apoB20.1. Mean residue ellipticity as a function of wavelength was recorded at 25°C at a protein concentration of 1 μM apoB19 (•) or apoB20.1 (▽).

Fig. 3.

Thermal denaturation of apoB19 and apoB20.1. Mean residue ellipticity at 222 nm of 1 μM solutions of apoB19 (dashed line) and apoB 20.1 (solid line) was sampled at two second intervals as the temperature was raised from 25 to 75°C. The lines are sigmoidal curve fits of the data. Inset: Van't Hoff plots; the solid lines are linear curve fits of the thermal denaturation data. Intercepts of the lines at the X axis yield the reciprocal of the transition temperature midpoint.

Fig. 4.

Fluorescence denaturation of apoB19 and apoB20.1. The wavelength of maximum fluorescence of 1 μM solutions of apoB19 (•) and apoB20.1 (▽) was monitored as a function of guanidine hydrochloride (GdnHCl) concentration.

Fig. 5.

Interfacial exclusion pressure of apoB19 and apoB20.1. ApoB19 (•) and apoB20.1 (▽) were injected beneath EPC monolayers spread at increasing initial surface pressures, and the resulting change in surface pressure (ΔP) was determined. The solid lines are linear regressions of the data. Extrapolation of the lines to the X axis yields exclusion pressures of 22.8 mN/m for apoB19, and 25.1 mN/m for apoB20.1.

Fig. 6.

Dynamic interfacial behavior of apoB19 and apoB20.1 at the oil/water interface. The binding of apoB19 and apoB20.1 to the triolein/water interface was measured using an ITC Tracker oil drop tensiometer. Ten μl drops of triolein were rapidly formed into buffer containing 25 μg/ml apoB19 (dashed line) or apoB20.1 (solid line), and the surface tension was continuously monitored.

Fig. 7.

Surface tension-area plots of apoB19 and apo 20.1 at the triolein/water interface. Ten μl drops of triolein were formed into buffer containing 25 μg/ml apoB19 or apoB20.1, as indicated. After the tension had stabilized, the drop volume was sinusoidally oscillated at 6 cycles/min and the change in surface tension was continuously monitored as a function of drop surface area.

Fig. 8.

Dynamic interfacial behavior of apoB19 at the EPC/triolein/water interface. A 10 μl triolein drop was rapidly formed into buffer (arrow a), and was coated with a phospholipid (PL) monolayer by addition of Intralipid (arrow b). Excess unbound PL was washed out of the cuvette by continuous buffer exchange (arrow c). Then 15 μg/ml apoB19 was added to the cuvette (arrow d) and the further decrease in interfacial tension was monitored. The elasticity of the interface at each stage was determined by sinusoidal oscillation of the drop volume (denoted by the rapid vertical displacements) as described under EXPERIMENTAL PROCEDURES. Analyses of apoB20.1 under the same conditions were also performed (36); data not shown.