UBC Theses and Dissertations
A fluorescence study of the COOH-terminus region of equine platelet tropomyosin Clark, Ian David
The use of fluorescent molecules as probes of protein conformation is recognized as a technique which provides very specific information and has been applied, in recent years, to the study of the role of tropomyosin (TM) in the regulation of contractile processes. The isolation and sequencing of TM from horse blood platelets (P-TM) has shown it to be different from muscle TM, especially near the NH₂-and COOH-termini. These differences have been suggested to weaken end-to-end interaction of P-TM molecules. TM's are two chain coiled coils and P-TM has cysteine residues at the penultimate COOH-terminus position of adjacent chains. These can be labelled with sulfhydryl-specific fluorescent probes that reflect conformational changes in that region of the molecule via changes in their emission characteristics. The results of experiments on both pyrene (Py) (40) and acrylodan (AD) labelled P-TM show that there is a preferred interaction of the COOH-terminus of P-TM with the NH₂-terminus of cardiac TM over that with the NH₂-terminus of P-TM. This indicates that the altered NH₂-terminus of P-TM, with respect to muscle TM, is responsible for the relative loss of polymerizability of P-TM at low salt concentration. Addition of actin to the Py-P-TM (40) and AD-P-TM species showed changes in emission characteristics indicative of binding to the F-actin filaments, suggesting that the presence of the probes had not affected the function of the P-TM adversely. However, the presence of pyrenes at the COOH-terminus seemed to reduce further the ability of P-TM to self-polymerize. Thermal denaturation of AD-P-TM, AD-C-TM and AD-labelled truncated P-TM followed by fluorescence polarization suggested that, contrary to the theory of Skolnick and Holtzer on the stability of two chain coiled coils, the region towards the COOH-terminus is among the last to lose its helical character.
Item Citations and Data