Probing the Lipid-Free Structure and Stability of Apolipoprotein A-I by Mutation
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- 作者：Irina N. Gorshkova ; Kalliopi Liadaki ; Olga Gursky ; David Atkinson ; and Vassilis I. Zannis
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：December 26, 2000
- 年：2000
- 卷：39
- 期：51
- 页码：15910 - 15919
- 全文大小：144K
- 年卷期：v.39,no.51(December 26, 2000)
- ISSN：1520-4995
文摘
To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free humanapolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specificmutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescencespectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering thesecondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triplesubstitution (L222K, F225K, F229K) leads to a small increase in the 
-helical content and stability,indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. TheC-terminal truncation 
(209-243) does not change the -helical content but reduces the protein stability.Truncation of a larger segment, (185-243), does not affect the secondary structure or stability. In contrast,an intermediate truncation, (198-243), leads to a significant reduction in the -helical content, stability,and unfolding cooperativity. The internal 11-mer deletion (187-197) has no significant effect on theconformation or stability, whereas another internal 11-mer deletion, (165-175), dramatically disruptsand destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial forproper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminalregion 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactionsin the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located inthe N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to theN-terminus.
-helical content and stability,indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. TheC-terminal truncation (209-243) does not change the -helical content but reduces the protein stability.Truncation of a larger segment, (185-243), does not affect the secondary structure or stability. In contrast,an intermediate truncation, (198-243), leads to a significant reduction in the -helical content, stability,and unfolding cooperativity. The internal 11-mer deletion (187-197) has no significant effect on theconformation or stability, whereas another internal 11-mer deletion, (165-175), dramatically disruptsand destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial forproper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminalregion 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactionsin the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located inthe N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to theN-terminus.