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UBC Theses and Dissertations

Coupled structural responses in tropomyosin Clark, Ian David


Fluorescence spectroscopy can be used to probe protein conformation and is recognized as a technique that provides very specific information. It has been applied/ in recent years/ to the study of tropomyosin (TM) and its role in regulation of contractile processes. In this thesis, two different approaches were used to further the understanding of the structure/function relationship in the two chain coiled coil of tropomyosin. The first involves a comparative study on TM and non-polymerizable TM (NPTM) (Mak, A.S., and Smillie, L.B. (1981) Biochim. Biophys. Res. Commun., 101, 208-214). Fluorescence involving pyrene (Py) and acrylodan (AD) bound at the only cysteine residue in the molecule (Cys-190), and circular dichroism (CD) studies led to the main conclusion that, while the two species, are very similar in stability, the COOH-terminus is required to hold the Cys-190 region in a specific conformation. This long-range structural effect may play a role in regulation of contraction. A species having one intact COOH-terminus, made by hybridizing TM and NPTM, was found to be non-polymerizable suggesting that one intact COOH-terminus is insufficient to permit overlap with the NH₂-terminus of a neighbouring TM under polymerizing conditions. Unlike the TM/NPTM hybrid, the hybrid of TM and platelet TM (P-TM) was difficult to make due to the sequence mismatches in the terminal regions, but small quantities could be detected by loss of excimer fluorescence from Py-P-TM on rapid cooling of a heated mixture of Py-P-TM and cardiac TM (C-TM). The second approach was to investigate the effect of actin-binding proteins on the structure and function of tropomyosin. DNase I depolymerizes F-actin and is known to interfere with the end-to-end polymerizability of tropomyosin (Payne, M.R., Baydoyannis, H., and Rudnick, S.E. (1986) Biochim. Biophys. Acta 883, 454-459). Results presented here from fluorescence studies suggest that this effect is caused by a localized loss of structure in the tropomyosin at the sites of labelling upon binding of DNase I. This result is supported by CD studies on labelled and unlabelled tropomyosins. Gelsolin is another actin-binding protein found in many cell types and in extracellular fluids. It is shown here to be able to depolymerize tropomyosin, but its mechanism of action is not the same as that of DNase I. The effect of interaction of gelsolin on the structure of tropomyosin, as determined from fluorescence studies, is negligible.

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