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 Tm,WT = 50 ± 2 °C and van't Hoff enthalpy ΔHv,WT(Tm) = 18 ± 2 kcal/mol. G15A substitution leads to an increased α-helical content of 42 ± 4% and an increased Tm,G15A = 57 ± 2 °C, which corresponds to stabilization by δΔGapp = +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.