Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe.
Apolipoprotein A-I (apoA-I) has important physiologic roles in reverse cholesterol transport, as a component of HDL; however, apoA-I also exists in lipid-poor or lipid-free forms that are key intermediates in HDL metabolism and acceptors of lipids from cells. The aim of this study was to examine the structure and stability of the central and C-terminal regions of lipid-free apoA-I. To this end, five Cys mutants of proapoA-I were constructed and expressed in Escherichia coli: V119C, A124C, A154C, A190C, and A232C. These mutants were specifically labeled with 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AC) and were examined by CD spectroscopy and a variety of fluorescence methods. The results showed that the introduction of Cys residues and their covalent labeling with AC did not affect the overall structure and stability of apoA-I. However, AC fluorescence properties revealed that different segments of the central and C-terminal half of apoA-I have distinct folding and stability properties. From fluorescence energy transfer data, average distances between the N-terminal region containing Trp residues and the various AC locations were obtained. The current results, together with previously published observations, led to the construction of a three-dimensional model for the folding of lipid-free apoA-I.
Behling Agree
Andrea K
AK
Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, University of Illinois, 506 South Mathews Avenue, Urbana, IL 61801, USA.
Tricerri
M Alejandra
MA
Arnvig McGuire
Kirsten
K
Tian
Shao-min
SM
Jonas
Ana
A
D015081:2-Naphthylamine / Q000031:analogs & derivatives*; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics; D001054:Apolipoproteins A / Q000096:biosynthesis / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D008081:Liposomes; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017510:Protein Folding; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D005456:Fluorescent Dyes; D008081:Liposomes; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C038518:acrylodan; D015081:2-Naphthylamine; D004134:Dimyristoylphosphatidylcholine