Abstract
Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.
Highlights
Excretion into the intestine as bile [1]
Site-directed spin labeling of Apolipoprotein A-I (apoA-I) followed by electron paramagnetic resonance (EPR) spectroscopy was used to analyze the structure of the N-terminal domain of apoA-I on reconstituted nascent HDL (rHDL)
The 93 spinlabeled apoA-I variants were converted to 9.6 nm rHDL, prepared by combining spin-labeled proteins with POPC lipids and cholesterol at a protein to lipid molar ratio of 1:4:80, as described under “Experimental Procedures.” rHDL particles with a diameter of 9.6 nm were isolated by size exclusion chromatography purification, as described previously [34]
Summary
Excretion into the intestine as bile [1]. High density lipoproteins (HDL) are the primary mediator of reverse cholesterol transport [2]. By electron paramagnetic resonance (EPR) spectroscopy analysis, we have determined the location of this “hinge domain” as a 12-amino acid-long segment centered on residue 139 Because this portion of apoA-I aligns with its counterpart in a paired apoA-I on HDL, we hypothesized this stretch of residues forms a pore-like structure, described in the “looped belt” model [14], wherein this pore provides lecithin:cholesterol acyltransferase access to cholesterol and the acyl chain of POPC. This has been supported by molecular dynamic simulation computational analysis, wherein Jones et al [23] determined that this region could form an amphipathic presentation tunnel for the acyl chains of POPC. Jones et al [23] observed that the sn-2 acyl chain of POPC, the preferred acyl chain that is transacylated to cholesterol by lecithin:cholesterol acyltransferase [24], can insert into the tunnel at a significantly higher frequency than the sn-1 acyl chain, further substantiating a functional role for this apoA-I conformational feature on HDL
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