title: Probing the lipid-free structure and stability of apolipoprotein A-I by mutation.
abstract: To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the alpha-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation Delta(209-243) does not change the alpha-helical content but reduces the protein stability. Truncation of a larger segment, Delta(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, Delta(198-243), leads to a significant reduction in the alpha-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion Delta(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, Delta(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.
authors: Section of Molecular Genetics at Whitaker Cardiovascular Institute and Department of Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA. igorshko@bu.edu
authors: I N
authors: IN
authors: Gorshkova
authors: K
authors: K
authors: Liadaki
authors: O
authors: O
authors: Gursky
authors: D
authors: D
authors: Atkinson
authors: V I
authors: VI
authors: Zannis
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D019791:Guanidine; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014407:Tumor Cells, Cultured
chemical_list: D016632:Apolipoprotein A-I; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019791:Guanidine