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Apolipoprotein (apo) A-V is a 343-residue, multidomain protein that plays an important role in regulation of plasma triglyceride homeostasis. Primary sequence analysis revealed a unique tetraproline sequence (Pro293-Pro296) near the carboxyl terminus of the protein. A peptide corresponding to the 48-residue segment beyond the tetraproline motif was generated from a recombinant apoA-V precursor wherein Pro295 was replaced by Met. Cyanogen bromide cleavage of the precursor protein, followed by negative affinity chromatography, yielded a purified peptide. Nondenaturing polyacrylamide gel electrophoresis verified that apoA-V(296-343) solubilizes phospholipid vesicles, forming a relatively heterogeneous population of reconstituted high-density lipoprotein with Stokes’ diameters>17 nm. At the same time, apoA-V(296-343) failed to bind a spherical lipoprotein substrate in vitro. Far-UV circular dichroism spectroscopy revealed the peptide is unstructured in buffer yet adopts significant R-helical secondary structure in the presence of the lipid mimetic solvent trifluoroethanol (TFE; 50% v/v). Heteronuclear multidemensional NMR spectroscopy experiments were conducted with uniformly 15N- and 15N/13C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOSþ reveal the peptide adopts R-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296-343) adopts an extended amphipathic R-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V.


Copyright © 2010 American Chemical Society. The definitive version is available at http://dx.doi.org/10.1021/bi1005859.

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