Our goal is to use the fibrous protein tropomysin (which in the native state is a two-chain, completely alpha-helical coiled-coil) as a natural model system to answer structural questions about associated alpha-helices. Specifically, we propose: 1) To measure rather directly, the energetics and specificity of the inter-helix interaction by measurement of the relative population of hybrid species (i.e., hetero-helicsal molecules in which two, unlike alpha-helical chains are associated) existing in equilibrium with the corresponding homo-helical species. Appropriate methodology has been developed in the previous grant period. This provides the equilibrium constant and therefore the standard free energy change of the hybridization reaction; the temperature dependence allows decomposition into enthalpy and entropy. With knowledge of the primary structures of both chains, these data may be interpretable in terms of specific, inter-helix, side-chain contacts. 2) To examine the thermally-induced double-helix to random-coil transitions over the entire accessible range of temperature by CD in the backbone region and by light-scattering. Studies in the previous period revealed that there are three, distinct cooperative transitions observable. Though comparison of these transitions as observed in the two homohelical species and the isolated hybrid species, it may be possible to assign these transitions to particular regions along the chain. Theories exist with which to compare these assignments. 3) To study the thermally induced alpha-helix to random-coil transitions in detergent (dodecyl sulfate) solutions, wherein the tropomyosin exists as spearated, partially alpha-helical chains. A rather successful theory has been used to obtain such properties as helix content (from primary structure) in presence and absence of dodecyl sulfate. Comparison may tell us what influcence the second helix has on the helix content of the first. 4) To introduce 13C-enriched labels at specific sites on both types of tropomyosin chains and follow the thermal transitions by 13C NMR. This should aid in the assignment of regions of the chain to specific thermal transitions and provide a probe of local motions at those sites.
