The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin.
We have used a synthetic peptide consisting of the first 30 residues of striated muscle alpha-tropomyosin, with GlyCys added to the C-terminus, to investigate the effect of N-terminal acetylation on the conformation and stability of the N-terminal domain of the coiled-coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low alpha-helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N-terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2-chained coiled coil at high ionic strength. Disulfide crosslinking and N-terminal acetylation both greatly stabilized the coiled-coil alpha-helical conformation in high salt. Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its alpha-helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled-coil formation. In the presence of the organic solvents, N-terminal acetylation had very little effect on the conformation or stability of the peptide. Our results indicate that N-terminal acetylation stabilizes coiled-coil formation in the peptide. The effect cannot be explained by interactions with the "helix-dipole" because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N-terminal acetylation has only small effects on the overall stability of tropomyosin.
Greenfield
N J
NJ
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635.
Stafford
W F
WF
Hitchcock-DeGregori
S E
SE
D000107:Acetylation; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D012997:Solvents; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics
D010446:Peptide Fragments; D012997:Solvents; D014335:Tropomyosin