Interfacial Exclusion Pressure Determines the Ability of Apolipoprotein A-IV Truncation Mutants to Activate Cholesterol Ester Transfer Protein

Abstract

We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer ␣-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the ␣-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic ␣-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r ‫؍‬ 0.790, p ‫؍‬ 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange.

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

“…Physiologically, proteins with higher ⌸ EX are better able to bind to and regulate the metabolism of lipoproteins with high local surface pressures, such as nascent HDL or catabolic products of TAG-rich lipoproteins ( 51 ). We hypothesized that greater ␣ -helical propensity would favor apoC-I binding to lipid interfaces, refl ected in larger ⌸ EX .…”

“…Exclusion pressure ( ⌸ EX ) for a peptide is the ⌸ at which the peptide is excluded from POPC/TO/W interfaces (i.e., it cannot penetrate into the interface to raise surface pressure) ( 43,51 ). Repetition of the ramping protocol over various ⌸ i gave different ⌬ ⌸ values for each ⌸ i .…”

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

“…Thermal denaturation studies were performed by monitoring ellipticity at 222 nm at 2 s intervals as the temperature of the cuvette was increased from 25°C to 75°C at 1°C/min. The enthalpy of denaturation, DH D , and thermal denaturation midpoint, T m , were determined from Vanʼt Hoff plots of DG vs. 1/T (38). The entropy of folding, DS, was calculated at the denaturation midpoint using the Gibbs-Helmholtz equation (38,39).…”

“…The enthalpy of denaturation, DH D , and thermal denaturation midpoint, T m , were determined from Vanʼt Hoff plots of DG vs. 1/T (38). The entropy of folding, DS, was calculated at the denaturation midpoint using the Gibbs-Helmholtz equation (38,39). Mean residue ellipticity at 222 nm, [Q] 222 , was calculated as ([Q] raw 3 MRW)/(10 3 l 3 C), where l is the path length in cm and C is the concentration in g/ml, using a mean residue weight (MRW) of 111.9 for apoB19 and 111.6 for apoB20.1.…”

“…Adsorption of apoB19 and apoB20.1 to the PL/water interface was examined using a KSV 5000 Langmuir film balance (KSV Instruments, Helsinki, Finland) (38,42). EPC monolayers were spread over 5.65 mM Na 2 HPO 4 , 3.05 mM NaH 2 PO 4 , 80 mM NaCl, pH 7.0 and allowed to stabilize.…”