Goonasekara, Charitha Lakshini (2008) Structure-function of native and amino-terminally truncated striated muscle tropomyosins. Doctoral (PhD) thesis, Memorial University of Newfoundland.
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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 truncated system (+Ca2+, ionic strength, 22mM) activates myosin to a greater extent than native tropomyosin, and has stronger affinity for myosin-Sl-ADP (+EGTA; ionic strength, 70mM), the reverse of what has been observed with the carboxy-terminally-shortened protein. These results suggest that the amino-terminal region of tropomyosin is an inhibitory thin filament element, which stabilizes the off-state of the thin filament via an interaction with the tail section of troponin-T, and that the two ends at the overlap complex have opposing regulatory roles. -- Finally, the conformational stability of tropomyosins from Atlantic salmon was characterized under a variety of conditions (salt, pH and osmolyte). The most variable sections of sequence encompass residues 9-49, 73-87 and 172-216. In two of these hotspots there is a pair of closely-spaced glycines, namely at residues 24 and 27 in fast skeletal tropomyosin and residues 83 and 87 in cardiac tropomyosin. Further, the isoform from fast muscle does not have a d position alanine between residues 172-216 due to the isomorphism Alal79Thr and thus lacks the fifth alanine cluster. These substitutions have reduced the conformational stability of the fish protein compared to the mammalian counterpart and provide insight into the adaptation of tropomyosin to cold temperature.
|Item Type:||Thesis (Doctoral (PhD))|
|Additional Information:||Bibliography: leaves 205-252|
|Department(s):||Science, Faculty of > Biochemistry|
|Library of Congress Subject Heading:||Amino acids; Hydrolysis; Tropomyosins--Structure|
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