CD and C-13(alpha)-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of alpha-tropomyosin

Synthesis and CD and C-13(alpha)-NMR studies in a near-neutral saline buffe r are reported for a 65-residue peptide (Tm-190(254)) comprising residues 1 90-254 of the alpha -tropomyosin chain. CD on a version disulfide cross-lin ked via the N-terminal cysteine side chains indicates that this dimer is hi ghly helical and melts near 48 degreesC. The CD is independent of peptide c oncentration, showing that association of Tm-190(254) stops at the two-stra nd level. Similar studies on the reduced version show, much lower helix con tent at low temperature, melting points below room temperature, and the exp ected concentration dependence. The observed melting temperature of the red uced peptide is far below (by 27 degreesC) that expected from an extant ana lysis of calorimetry data on parent tropomyosin that designates Tm-190(254) as an independently melting "cooperative block." This disagreement and the pronounced nonadditivity seen when data for Tm-190(254) are combined with extant data for other subsequences argue decisively against the concept of specific independently melting blocks within the tropomyosin chain. The dat a for Tm-190(254) also serve to test recent ideas on the sequence determina nts of structure and stability in coiled coils. Analysis shows that some id eas, such as the stabilizing effect of leucine in the d heptad position, fi nd support, but others-such as the destabilizing effect of alanine in d, th e dimer-disfavoring effect beta -branching in d and its dimer-favoring effe ct in a, and the dimer-directing effect of asparagine in a-are more questio nable in tropomyosin than in the leucine zipper coiled coils. C-13(alpha)-N MR data at two labeled sites, L228(d) and V246(a), of Tm-190(254) display w ell-separated resonances for folded and unfolded forms at each site, indica ting that the transition is slow on the NMR time scale and thus demonstrati ng the possibility of obtaining thermodynamic and kinetic information on th e transition at the residue level. (C) 2001 John Wiley & Sons, Inc.