Synthetic model for two-stranded alpha-helical coiled-coils. Design, synthesis, and characterization of an 86-residue analog of tropomyosin.
A 43-residue peptide analog of tropomyosin Ac-AB4C-OH (A = Lys-Cys-Ala-Glu-Leu-Glu-Gly, B = Lys-Leu-Glu-Ala-Leu-Glu-Gly, C = Lys-Leu-Glu-Ala-Leu-Glu-Gly-Lys) was synthesized. The 86-residue disulfide-linked dimer was prepared by air oxidation of the single cysteine residue in the NH2-terminal Fragment A of the 43-residue peptide to provide a two-stranded alpha-helical coiled-coil of defined molecular weight with the chains in-register and parallel. The physical properties of the 86-residue dimer were determined and compared to CM-tropomyosin and sequential polyheptapeptides. The stabilizing effect of the disulfide bridge in the synthetic dimer was indicated by the shift in the transition of the thermal unfolding profile (t 1/2) of +6.5 degrees C from 72.5 degrees C for the reduced sample to 79 degrees C for the oxidized sample. The 86-residue disulfide-linked dimer maintains 65% of the original helicity at 65 degrees C, and the polyheptapeptide [Leu-Glu-Serr-Leu-Glu-Ser-Lys]n of 9,500 daltons in denaturant maintains 75% helicity at 65 degrees C, whereas CM-tropomyosin is completely denatured at this temperature. These results show a shift in the transition of the thermal unfolding profile (t 1/2) of greater than 39 degrees C for the above two synthetic peptides relative to CM-tropomyosin. The polyheptapeptide [Leu-Glu-Ser-Leu-Glu-Ser-Lys]n maintains 95% of the original helicity in 3 M urea, whereas 50% remains in CM-tropomyosin. Comparison of the CD spectra of three polyheptapeptides, [Leu-Glu-Ser-Leu-Glu-Ser-Lys]n, [Ala-Glu-Ser-Leu-Glu-Ser-Lys]n, and [Ala-Glu-Ser-Ala-Glu-Ser-Lys]n showed that the ellipticities increased as the size of the hydrophobic side chains increased in the positions responsible for the formation and stabilization of the coiled-coil. That substituting residues in the outer positions of the coiled-coil can affect alpha-helix stabilization is shown by comparing the CD spectra of three polymers [Leu-Glu-Ser-Leu-Glu-Ser-Lys]n, [Leu-Glu-Ser(Ac)-Leu-Glu-Ser(Ac)-Lys]n, and [Leu-Glu-Ala-Leu-Glu-Ala-Lys]n.
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides; D004229:Dithiothreitol; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000138:chemical synthesis; D011487:Protein Conformation; D011489:Protein Denaturation; D014335:Tropomyosin / Q000138:chemical synthesis
C028363:86-residue tropomyosin; D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014335:Tropomyosin; D004229:Dithiothreitol