THE STRUCTURE OF THE N-TERMINUS OF STRIATED-MUSCLE ALPHA-TROPOMYOSIN IN A CHIMERIC PEPTIDE - NUCLEAR-MAGNETIC-RESONANCE STRUCTURE AND CIRCULAR-DICHROISM STUDIES

Tropomyosins (TMs) are highly conserved, coiled-coil, actin binding re gulatory proteins found in most eukaryotic cells. The amino-terminal d omain of 284-residue TMs is among the most conserved and functionally important regions. The first nine residues are proposed to bind to the carboxyl-terminal nine residues to form the ''overlap'' region betwee n successive TMs, which bind along the actin filament. Here, the struc ture of the N-terminus of muscle alpha-TM, in a chimeric peptide, TMZi p, has been solved using circular dichroism (CD) and two-dimensional p roton nuclear magnetic resonance (2D H-1 NMR) spectroscopy. Residues 1 -14 of TMZip are the first 14 N-terminal residues of rabbit striated a lpha-TM, and residues 15-32 of TMZip ate the last 18 C-terminal residu es of the yeast GCN4 transcription factor. CD measurements show that T MZip forms a two-stranded coiled-coil or-helix with an enthalpy of fol ding of -34 +/- 2 kcal/mol. In 2D(1)H NMR studies at 15 degrees C, pH 6.4, the peptide exhibits 123 sequential and medium range intrachain N OE cross peaks per chain, characteristic of alpha-helices extending fr om residue 1 to residue 29, together with 85 long-range NOE cross peak s arising from interchain interactions. The three-dimensional structur e of TMZip has been determined using these data plus an additional 509 intrachain constraints per chain. The coiled-coil domain extends to t he N-terminus. Amide hydrogen exchange studies, however, suggest that the TM region is less stable than the GCN4 region. The work reported h ere is the first atomic-resolution structure of any region of TM and i t allows insight into the mechanism of the function of the highly cons erved N-terminal domain.