The carboxyl-terminal segment of apolipoprotein A-V undergoes a lipid-induced conformational change.
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 alpha-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 (15)N- and (15)N/(13)C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOS+ reveal the peptide adopts alpha-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296-343) adopts an extended amphipathic alpha-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V.
Mauldin
Kasuen
K
Center for Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, California 94609, USA.
Lee
Brian L
BL
Oleszczuk
Marta
M
Sykes
Brian D
BD
Ryan
Robert O
RO
D000595:Amino Acid Sequence; D000072040:Apolipoprotein A-V; D001054:Apolipoproteins A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism / Q000502:physiology*; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics
C437704:APOA5 protein, human; D000072040:Apolipoprotein A-V; D001054:Apolipoproteins A; D008051:Lipid Bilayers; D008075:Lipoproteins, HDL; D010446:Peptide Fragments; D011392:Proline; D008715:Methionine
J Lipid Res. 2008 Mar;49(3):572-80
18056685
Science. 1991 May 24;252(5009):1162-4
2031185
J Biomol NMR. 2009 Aug;44(4):213-23
19548092
Science. 2001 Oct 5;294(5540):169-73
11588264
Prog Lipid Res. 2004 Jul;43(4):350-80
15234552
Clin Chem. 2005 Feb;51(2):351-9
15528295
Arterioscler Thromb Vasc Biol. 2005 Feb;25(2):411-7
15591215
Protein Expr Purif. 2003 Jan;27(1):98-103
12509990
FEBS Lett. 1993 Jan 18;316(1):27-33
8422936
J Lipid Res. 2009 Apr;50 Suppl:S150-5
19050314
J Biomol NMR. 1994 Sep;4(5):603-14
22911360
J Biol Chem. 1996 Oct 25;271(43):26855-62
8900168
Biochemistry. 2008 Aug 19;47(33):8768-74
18652480
Curr Opin Lipidol. 2007 Jun;18(3):319-24
17495607
Biochemistry. 2007 Mar 27;46(12):3896-904
17326667
Biochemistry. 2003 Aug 12;42(31):9416-23
12899628
J Biol Chem. 2005 Jun 3;280(22):21553-60
15774484
Methods Enzymol. 1986;128:417-31
3724517
J Clin Invest. 2005 Oct;115(10):2862-9
16200213
J Biomol NMR. 1995 Nov;6(3):277-93
8520220
J Biol Chem. 2007 May 25;282(21):15484-9
17401142
Biophys J. 1967 Mar;7(2):121-35
6048867
J Biol Chem. 2009 Nov 27;284(48):33369-76
19825998
J Biol Chem. 2005 Jul 8;280(27):25383-7
15878877
Biochemistry. 1998 Jul 14;37(28):10203-10
9665727
J Lipid Res. 2009 Aug;50(8):1548-55
19098282
J Biomol NMR. 2008 Jan;40(1):31-48
17985196
J Biol Chem. 2001 Nov 30;276(48):44512-20
11577099