Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Living organisms move. The most spectacular examples are muscles that have a striped appearance under the microscope. We rely on them daily for blood circulation, posture, speed & strength. These basics of life arise from the interplay of motion-producing (myofilament) proteins. One of these, tropomyosin, is the focus of the application.

What does it do? Tropomyosin turns muscle on and off. It is essential because regulation is essential. Without it, a muscle would be like a uncontrollable vehicle, one lacking an ignition and brakes. When Ca(II) is released from internal stores, tropomyosin instigates a structural rearrangement of the thin filament, which then activates the motor myosin. The outcome is substantial ATP hydrolysis and contraction. When Ca(II) is taken back up, the reverse happens and relaxation ensues. Tropomyosin is able to do this because: (a) it polymerises with itself, forming a flexible, continuous strand and (b) its interactions with other proteins (eg actin, troponin) are Ca(II)-sensitive.

Part (I). 'Tuning' tropomyosin. The diversity of animal locomotion is an outcome of different types of muscle - fast, slow and cardiac - that deploy isoforms of the myofilament proteins to contract and relax. Protein isoforms are like cousins; they bear a family resemblance but have distinguishing features (eg slightly different amino acid sequences). How tropomyosin contributes to muscle diversity has long-been an enigma.

The major isoforms of tropomyosin are Tpm1.1 (formerly α) & Tpm2.2 (formerly β). They share 39 amino acid substitutions. The 'big' Q. What do they do differently? was answered only recently (Lohmeier-Vogel & Heeley 2016). Differences in polymerisation, troponin binding & regulation of myosin ATPase were reported. The 2016 study establishes a foundation to ask the next Q. Which isomorphisms determine tropomyosin isotype? Isomorphisms will be interchanged between Tpm1.1 and 2.2 with the intent of characterising the role of a particular isomorphism. Part (I) is also of relevance to disease mutations (currently > 50) not yet studied at the protein level.

Part (II). Tropomyosin in extreme conditions The problem of cold for a protein is rigidity. During the winter, the N. Atlantic approaches freezing, a drop of > 30 o C compared to the warm-blooded (mammalian) scenario. Yet this is a natural marine habitat. A 'big' Q. is: How does tropomyosin avoid being ‘straitjacketed’? Recent work (Fudge & Heeley 2015) has shown the existence of 3 'flexing' strategies (fewer ion pairs, more glycine & polar residues in core sites) within salmon Tpm1.1. The effect of these 'cold isomorphisms' on salmon Tpm1.1 function - polymerisation, troponin binding & regulation - will be probed by interchange of amino acids with the thermally-stable mammalian homologue. Part (II), as with Part (I), will provide information on tropomyosin's functional landscape (ie binding sites).