title: Limited proteolysis and biophysical characterization of the lipovitellin homology region in apolipoprotein B.
abstract: Apolipoprotein B (apoB) is the essential nonexchangeable protein in chylomicrons and very low-density lipoprotein-derived lipoprotein particles, including low-density lipoprotein (LDL). ApoB has been a key target for cardiovascular research because of its essential role in the assembly, secretion, delivery, and receptor binding of LDL. The three-dimensional structure of apoB has not been determined. However, the N-terminal region of apoB is homologous to the lipid storage protein lipovitellin, which allows the modeling of this region based on the X-ray structure of lipovitellin. The model of the N-terminal 17% of apoB (B17) suggests that, like lipovitellin, B17 consists of an N-terminal beta-barrel domain, a helical domain, and a beta-sheet domain (C-sheet). Here we test the validity of this model by limited proteolysis of B17 and the characterization of individual domains expressed in Escherichia coli and insect cell systems that are consistent with the model and proteolysis data. Circular dichroism studies of the individual domains indicate that they are folded and their secondary structures are in agreement with the model. We find that the helical domain and C-sheet of apoB interact with each other in vitro, suggesting a strong interaction between these two domains, even without a covalent peptide bond linkage. Our data suggest that the three lipovitellin-like domains exist in B17. Furthermore, the domains fold independently with secondary structures and stabilities like those of intact B17.
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA.
authors.forename: Zhenghui Gordon
authors.initials: ZG
authors.lastname: Jiang
authors.forename: Margaretha
authors.initials: M
authors.lastname: Carraway
authors.forename: C James
authors.initials: CJ
authors.lastname: McKnight
mesh_terms: D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001703:Biophysics / Q000379:methods; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004527:Egg Proteins; D015387:Egg Proteins, Dietary / Q000302:isolation & purification* / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000235:genetics; D014357:Trypsin / Q000378:metabolism*
chemical_list: D001055:Apolipoproteins B; D003432:Cross-Linking Reagents; D004527:Egg Proteins; D015387:Egg Proteins, Dietary; D010446:Peptide Fragments; C001549:lipovitellin; D014357:Trypsin