Abstract
We have used fluorescence and circular dichroism spectroscopy to investigate the effect of phospholipid on the structure and molecular stability of human apolipoprotein A-IV (apo-A-IV). Binding of apo-A-IV to egg phosphatidylcholine vesicles was rapid and did not cause release of encapsulated 6-carboxyfluorescein. Fluorometric titration established that apo-A-IV bound to the vesicles with an association constant of 1.36 x 10(6) liters/mol and a binding maximum of 2 molecules per vesicle. Binding of apo-A-IV to the vesicle surface caused a progressive increase in alpha helicity from 43% at baseline to 83% at saturation; denaturation studies showed that the free energy of stabilization of binding was 6.31 kcal/mol. Fluorescence quenching studies revealed that binding of apo-A-IV to the vesicles was associated with a dramatic decrease in the fractional exposure of tyrosine to iodide, and a decrease in the efficiency of intramolecular tyrosine-tryptophan energy transfer. These findings suggest that the binding of apo-A-IV to the vesicle surface may involve a relaxation of the globular protein conformation in which the tyrosine containing alpha-helical domains surrounding the tryptophan "unfold" and reorient their hydrophobic faces toward the phospholipid monolayer, with a consequent induction of additional alpha-helical structure. However, our data also suggest that apo-A-IV does not penetrate deeply into the region of the phospholipid fatty acyl chains, but rather sits higher in the monolayer, intercalated between the charged phospholipid head groups. This characteristic may determine the labile interaction of apo-A-IV with high density lipoproteins.
Highlights
These findings suggest that the binding of apo-A-IV to the vesicle surface may involve a relaxation of the globular protein conformation in which the tyrosine containing o-helical domains surrounding the tryptophan “unfold” and reorient their hydrophobic faces toward the phospholipid monolayer, with a consequent induction of additional a-helical structure
Rate and Affinity of Binding to Phospholipid Vesicles-The rate of binding of apo-A-IV to egg yolk phosphatidylcholine vesicles (EPCV) was examined by monitoring time-dependent changes in fluorescence intensity and circular dichroism
Total fluorescence intensity between 290 and 370 nm was monitored as aliquots of buffered 8 M guanidine hydrochloride were added to solutions of pure apo-A-IV (O), apo-A-IV-phospholipid-cholesterol micellar complexes (A), and apo-A-IV bound to EPCV (H)
Summary
A-IV-Human apo-A-IV was isolated from lipoprotein-depleted serum (d > 1.25 g/ml) by adsorption to a phospholipid-triglyceride emulsion followed by delipidation and anion exchange chromatography in 7.2 M urea [12]. The MC were concentrated by ultrafiltration in an Amicon 8050 cell using a YM-10 membrane and analyzed for protein [24], phospholipid [26], and cholesterol Analysis of the MC by gel filtration chromatography on a calibrated 50.cm Sepharose CL-4B column revealed a homogeneous population with an average Stokes radius of 8.8 nm and an apo-A-IV/phospholipid/cholesterol molar ratio of 1:496:12. Increasing concentrations of EPCV were added to solutions of apo-A-IV at two different concentrations (C, = 138 nM; C, = 550 nM), and the areas under the emission spectra from 300 to 370 nm, corrected for dilution, were determined. The fluorescence intensity at each phospholipid concentration was normalized as AI/AZ,,,,, (where AZ is the increase in intensity following each addition of EPCV, and AZ,,, is the difference between the maximum and base-line fluorescence intensity) and plotted against the phospholipid/apo-A-IV ratio.
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