Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide.
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 alpha-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% alpha-helical structure at 22 degrees C that melts reversibly with T(m,WT) = 50 +/- 2 degrees C and van't Hoff enthalpy deltaH(v,WT)(Tm) = 18 +/- 2 kcal/mol. G15A substitution leads to an increased alpha-helical content of 42 +/- 4% and an increased T(m,G15A) = 57 +/- 2 degrees C, which corresponds to stabilization by delta deltaG(app) = +0.4 +/- 1.5 kcal/mol. G15P mutant has approximately 20% alpha-helical content at 22 degrees C and unfolds with low cooperativity upon heating to 90 degrees C. R23P and T45P mutants are fully unfolded at 0-90 degrees 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 alpha-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 alpha-helical content reaching 74%. Comparison with the maximal alpha-helical content of 73% observed in lipid-bound apoC-1 suggests that the TMAO-stabilized secondary structure resembles the functional lipid-bound apolipoprotein conformation.
Gursky
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Department of Biophysics, Boston University School of Medicine, Massachusetts 02118-2526, USA. gursky@med-biophd.bu.edu
D019943:Amino Acid Substitution; D053302:Apolipoprotein C-I; D001056:Apolipoproteins C / Q000097:blood / Q000737:chemistry*; D002942:Circular Dichroism; D006801:Humans; D008744:Methylamines / Q000737:chemistry*; D015335:Molecular Probes; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet
D053302:Apolipoprotein C-I; D001056:Apolipoproteins C; D008744:Methylamines; D015335:Molecular Probes; C005855:trimethyloxamine