Probing the conformation of a human apolipoprotein C‐1 by amino acid substitutions and trimethylamine‐N‐oxide

Abstract To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine‐N‐oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C‐1 (apoC‐1, 57 residues) containing point mutations in the predicted α‐helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild‐type and plasma apoC‐1 in neutral low‐salt solutions superimpose, indicating 31 ± 4% α‐helical structure at 22 °C that melts reversibly with T m,WT = 50 ± 2°C and van't Hoff enthalpy Δ H v,WT ( T m ) = 18 ± 2 kcal/mol. G15A substitution leads to an increased α‐helical content of 42 ± 4% and an increased T m,G15A = 57 ± 2°C, which corresponds to stabilization by Δ G app = +0.4 ± 1.5 kcal/mol. G15P mutant has ∼∼20% α‐helical content at 22 °C and unfolds with low cooperativity upon heating to 90 °C. R23P and T45P mutants are fully unfolded at 0–90 °C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative α‐helical unit in apoC‐1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self‐associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil‐to‐helix transition in apoC‐1, with the maximal α‐helical content reaching 74%. Comparison with the maximal α‐helical content of 73% observed in lipid‐bound apoC‐1 suggests that the TMAO‐stabilized secondary structure resembles the functional lipid‐bound apolipoprotein conformation.