Madhushika M. Silva - November 25, 1029

How the protein tropomyosin increases its flexibility to adapt to cold marine environment.

Tropomyosin is an alpha helical coiled coil protein having a structural and regulatory role in the thin filament of skeletal and cardiac muscle. It is highly conserved. For example, tropomyosin from the major trunk muscle of Atlantic salmon (Salmo salar) shares just 20 amino acid differences (> 92% identity) with the homologue in mammals. Yet Salmo salar thrives at temperatures that are near freezing. How does the very same protein function optimally in two different temperatures? And how is salmon tropomyosin able to maintain a sufficient flexibility to function under such conditions? The answer must reside in the changes in its amino acid sequence. The basis of relative-instability of salmon tropomyosin was investigated by mutagenically reversing the only charge substitution; res 77 (Thr salmon; Lys rabbit), the only core substitution; res 179 (Ala salmon; Thr rabbit) and replacing a unique pair of glycines at res 24 & 27 with alanine.
The conformational stability of native and recombinant tropomyosins is investigated using calorimetry and limited chymotryptic digestion followed by Edman-sequencing of detected fragments. The interactions of tropomyosin with its thin-filament binding partners are investigated using F-actin co-sedimentation and troponin affinity chromatography.
It is found that, loss of an ionic interaction is a critical factor in destabilizing >150 AA stretch of the molecule, thereby increasing the flexibility of the coiled coil. Also, absence of an acetyl group (known to disrupt tropomyosin’s N-terminal region) dramatically weakens affinity for troponin. However, affinity is regained by removing the helix-breaking glycines. These findings demonstrate the existence of a troponin binding site at the N-terminal end of tropomyosin (in addition to known binding site at the C-terminal end) having possible regulatory significance.



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