Biochemical characterization of the roles of glycines 24 and 27 and threonine 179 in tropomyosin from the fast skeletal trunk muscle of the atlantic salmon.

Korrina R Fudge, David H Heeley

Biochemistry 2015 May 5

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Atlantic salmon fast skeletal muscle is composed of an α-type tropomyosin that shares 20 substitutions with a mammalian homologue. Prominent isomorphisms considered to be potentially destabilizing include threonine 179 (core, Ala in rabbit) and a unique pair of glycines, residues 24 and 27. Bacterially expressed mutant tropomyosins were isolated without exposure to elevated temperature or organic solvent. The Thr179Ala mutant is more resistant to heat-induced denaturation (Tm > 40 °C) than the nonmutant control (Tm = 37 °C) as monitored by far-UV circular dichroism [0.1 M salt and 1 mM DTT (pH 7)]. Changing one glycine to alanine has no detectable effect on the Tm versus the control value. An increase of 1 °C is observed for the double mutant (Gly27Ala/Gly24Ala). The preferred site of chymotryptic cleavage of recombinant salmon tropomyosin is between Leu 11 and Lys 12. The rate of the subsequent cleavage at Leu 169, the preferred site in rabbit tropomyosin, is reduced via replacement of Thr 179 with Ala. Thin filaments reconstituted with this mutant display greater Ca(II) induction of the myosin steady-state MgATPase versus control. Glycine-induced local instability is demonstrated by Escherichia coli outer membrane protease T (Omp-T) cleavage between Lys 6 and Lys 7. The control is more susceptible to proteolysis at this site than the mutants, which are distinct from each other: control > Gly27Ala > Gly24Ala > double mutant (most resistant to Omp-T). Similarly, the double mutant is comparatively more resistant to cleavage at Leu 11. By inference, the mid- and amino-terminal sections of salmon tropomyosin are intrinsically less stable than those in mammals, suggesting greater flexibility. Neighboring glycines make up a new destabilization motif in tropomyosin.

Citation

Korrina R Fudge, David H Heeley. Biochemical characterization of the roles of glycines 24 and 27 and threonine 179 in tropomyosin from the fast skeletal trunk muscle of the atlantic salmon. Biochemistry. 2015 May 5;54(17):2769-76