The N-terminus of apolipoprotein A-V adopts a helix bundle molecular architecture

Abstract

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic alpha-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein.

FIGURE 1

SDS-PAGE analysis of pepsin-limited proteolysis of full-length apoA-V. Proteins were electrophoresed on a 10 to 20% acrylamide-SDS slab gel and stained with Gel Code Blue reagent: lane 1, untreated full-length apoA-V; lane 2, molecular mass markers; lane 3, pepsin-treated full-length apoA-V at 0 min; lane 4, pepsin treated for 5 min; lane 5, pepsin treated for 30 min; lane 6, pepsin treated for 60 min; and lane 7, pepsin treated for 120 min. NT denotes the N-terminal fragment and CT the C-terminal fragment.

FIGURE 2

Helical wheel diagram for a predicted helix in apoA-V(1–146). The amino acid residues depicted are numbered with hydrophobic residues circled in bold and charged residues indicated.

FIGURE 3

Amino acid sequence of apoA-V(1–146). Residues postulated to adopt α-helical secondary structure, as predicted with Coils analysis, are underlined. Proline residues are depicted in bold.

FIGURE 4

SDS-PAGE analysis of isolated apoA-V truncation variants: lane 1, molecular mass markers; lane 2, full-length apoAV; lane 3, apoA-V(1–292); lane 4, apoA-V(1–169); and lane 5, apoA-V(1–146).

FIGURE 5

Guanidine HCl denaturation of apoA-V(1–146). The molar ellipticity of the protein was measured at 222 nm at a concentration of 0.2 mg/mL in 50 mM citrate and 150 mM NaCl (pH 3.0) (○) or 20 mM phosphate buffer (pH 7.4) (●), at various concentrations of guanidine HCl (± standard deviation; n = 4).

FIGURE 6

Effect of proteins on ANS fluorescence: (a) albumin, (b) apoA-V(1–146), (c) apoLp III, (d) apoE3 NT, and (e) ANS in buffer.

FIGURE 7

Trp fluorescence quenching of apoA-V(1–146) by KI at pH 7.4. Stern–Volmer plots of apoA-V(1–146) Trp5 (○), apoA-V(1–146) Trp97 (●), and apoA-V(1–146) Trp73 (□).