Combined N- and C-Terminal Truncation of Human Apolipoprotein A-I Yields a Folded, Functional Central Domain

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Abstract
A combined N- and C-terminal truncation variant of human apolipoprotein A-I (apoA-I) was designed, expressed in Escherichia coli, isolated, and characterized. Hydrodynamic experiments yielded a weight average molecular weight of 34000, indicating apoA-I-(44−186) exists in solution predominantly as a dimer. An axial ratio of 4.2 was calculated for the dimer based on sedimentation velocity experiments. Far-UV circular dichroism spectroscopy of apoA-I-(44−186) in buffer indicated the presence of 65% α-helix secondary structure. Guanidine hydrochloride denaturation experiments yielded a transition midpoint of 0.5 M for apoA-I-(44−186). ApoA-I-(44−186) induced solubilization of dimyristoylphosphatidylcholine vesicles at a rate comparable to that of full-length apoA-I, displayed lipoprotein binding ability, and was an acceptor of ABCA1-mediated cholesterol efflux from cultured macrophages. Fluorescence quenching studies with KI indicate that the three Trp residues in apoA-I-(44−186) are shielded from the aqueous environment. Taken together, the data indicate that lipid-free apoA-I-(44−186) adopts a folded conformation in solution that possesses lipid binding capability. The central region of apoA-I appears to adopt a globular amphipathic α-helix bundle organization that is stabilized by intramolecular and/or intermolecular helix−helix interactions. Lipid association likely results in a conformational adaptation wherein helix−helix contacts are substituted for helix−lipid interactions.