Probing the lipid-free structure and stability of apolipoprotein A-I by mutation

To probe the secondary structure of the C-terminus (residues 165-243) of li pid-free human apolipoprotein A-I (apoA-I) and its role in protein stabilit y, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescenc e spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 an d G186 are located in a loop/disordered region. A triple substitution (L222 K, F225K, F229K) leads to a small increase in the alpha -helical content an d stability, indicating that L222, F225, and F229 are not involved in stabi lizing hydrophobic core contacts. The C-terminal truncation Delta>(*) over bar * (209-243) does not change the a-helical content but reduces the prote in stability. Truncation of a larger segment, Delta>(*) over bar * (185-243 ), does not affect the secondary structure or stability. In contrast, an in termediate truncation, Delta>(*) over bar * (198-243), leads to a significa nt reduction in the alpha -helical content, stability, and unfolding cooper ativity. The internal 11-mer deletion Delta>(*) over bar * (187-197) has no significant effect on the conformation or stability, whereas another inter nal 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 finding s 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 sta bilizing interactions in the molecule. The effect of the substitution (G185 P, G186P) on the exposure of tryptophans located in the N-terminal half sug gests an apoA-I tertiary conformation with the C-teminus located close to t he N-terminus.