Structure-function of native and amino-terminally truncated striated muscle tropomyosins

• Main Author: Goonasekara, Charitha Lakshini
• Format: Thesis
• Language: English
• Published: Memorial University of Newfoundland 2008
• Subjects: Atlantic salmon
• Online Access: https://research.library.mun.ca/1645/; https://research.library.mun.ca/1645/1/Goonasekara_CharithaLakshini.pdf; https://research.library.mun.ca/1645/3/Goonasekara_CharithaLakshini.pdf

Tropomyosin is a polymeric protein found in the muscle thin filament together with actin and troponin. The end regions, which encompass the first and last 10-11 amino acids, are vital to its function. We have analyzed the role of the amino-terminal region by selective removal of the first six amino acids. A method describing the hydrolysis of the sixth peptide bond of tropomyosin by bacterial outer membrane protease T (Omp T) is described. -- Compared to the native counterparts, shortened forms of rabbit skeletal and Atlantic salmon fast skeletal tropomyosins (residues 7 - 284), as well as the unacetylated (residues 1 - 284) version of the latter, all display reduced affinity for both troponin and the amino-terminal fragment of rabbit skeletal troponin-T (N-Tn-T, residues 1 - 158), as judged by affinity chromatography. Thus, loss of the hexapeptide, together with the blocking group, weakens the binding of tropomyosin and troponin-T. Omp T-digested tropomyosin binds weakly to F-actin in the micromolar concentration range, but this interaction is restored by troponin. At moderate ionic strength (50mM KC1), the apparent Kds are: 0.26 uM (+EGTA) and 1.6 uM (+Ca2+). The inductive property of troponin is attributable, in part, to troponin-I. But whereas the amino-terminal fragment of troponin- T (residues 1-158) enhances troponin-I-induced F-actin binding of two other tropomyosin products which have weak affinity for F-actin, specifically, recombinant unacetylated tropomyosin (residues 1 - 284) and carboxypeptidase-digested tropomyosin (residues 1 - 273, Heeley, D. H. et al (1987) J. Biol. Chem. 262,9971-9978), it is ineffective in the case of the Omp T-digested protein. These data are evidence for a troponin-T binding site within the amino-terminal region of tropomyosin. -- In assays that contain myosin, thin filaments composed of Omp T-digested tropomyosin are less cooperative than native thin filaments, consistent with the disruption of physical continuity within the filament. At the same time, however, the ...