The N-terminus of apolipoprotein A-V adopts a helix bundle molecular architecture.
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.
Wong
Kasuen
K
Center for Prevention of Obesity, Diabetes and Cardiovascular Disease, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, California 94609, USA.
Beckstead
Jennifer A
JA
Lee
Dustin
D
Weers
Paul M M
PM
Guigard
Emmanuel
E
Kay
Cyril M
CM
Ryan
Robert O
RO
D000595:Amino Acid Sequence; D000072040:Apolipoprotein A-V; D001054:Apolipoproteins A / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
C437704:APOA5 protein, human; D000072040:Apolipoprotein A-V; D001054:Apolipoproteins A; D011994:Recombinant Proteins; D014364:Tryptophan