期刊
BIOCHEMISTRY
卷 39, 期 51, 页码 15910-15919出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi0014406
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资金
- NHLBI NIH HHS [HL 48739, P01HL 26335] Funding Source: Medline
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>(*) over bar * (209-243) does not change the a-helical content but reduces the protein stability. Truncation of a larger segment, Delta>(*) over bar * (185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, Delta>(*) over bar * (198-243), leads to a significant reduction in the alpha -helical content, stability, and unfolding cooperativity. The internal 11-mer deletion Delta>(*) over bar * (187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, Delta>(*) over bar * (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-teminus located close to the N-terminus.
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