The N-Terminus of Apolipoprotein A-V Adopts a Helix Bundle Molecular Architecture

• Published in: Biochemistry (Easton) 2008-08, Vol.47 (33), p.8768-8774
• Main Authors: Wong, Kasuen, Beckstead, Jennifer A, Lee, Dustin, Weers, Paul M. M, Guigard, Emmanuel, Kay, Cyril M, Ryan, Robert O
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Apolipoprotein A-V; Apolipoproteins A - chemistry; Apolipoproteins A - metabolism; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Sequence Data; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Spectrometry, Fluorescence; Tryptophan - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi800515c

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 α-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.