Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin.

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.

Conditions affecting production of functional muscle recombinant alpha-tropomyosin in Saccharomyces cerevisiae.

Yeasts are attractive hosts for heterologous protein production as they follow the general eukaryotic post-translational modification pattern. The well-known Saccharomyces cerevisiae has been used to produce a large variety of foreign proteins. The proper function of muscle tropomyosin depends on a specific modification at its N-terminus. Although tropomyosin has been produced in different expression systems, only the recombinant protein produced in the yeast Pichia pastoris has native-like functional properties. In this paper we describe the production of functional skeletal muscle tropomyosin in the yeast S. cerevisiae. The recombinant protein was produced in high amounts and production was strongly affected by genetic and environmental factors, including plasmid copy number, promoter strength, and growth media composition.

The cardiac mutant Mexican axolotl is a unique animal model for evaluation of cardiac myofibrillogenesis.

Hearts from cardiac mutant Mexican axolotl, Ambystoma mexicanum, do not form organized myofibrils and fail to beat. Though previous biochemical and immunohistochemical experiments showed a possible reduction of cardiac tropomyosin it was not clear that this caused the lack of organized myofibrils in mutant hearts. We used cationic liposomes to introduce both rabbit and chicken tropomyosin protein into whole hearts of embryonic axolotls in whole heart organ cultures. The mutant hearts had a striking increase in the number of well-organized sarcomeric myofibrils when treated with rabbit or chicken tropomyosin. FITC-labeled rabbit tropomyosin was used to examine the kinetics of incorporation of the exogenous protein into mutant hearts and confirmed the uptake of exogenous protein by the cells of live hearts in culture. By 4 h of transfection, both normal and mutant hearts were found to incorporate FITC-labeled tropomyosin into myofibrils. We also delivered an anti-tropomyosin antibody (CH 1) into normal hearts to disrupt the existing cardiac myofibrils which also resulted in reduced heartbeat rates. CH1 antibody was detected within the hearts and disorganization of the myofibrils was apparent when compared to normal controls. Introduction of a C-protein monoclonal antibody (ALD 66) did not result in a disruption of organized myofibrils. The results show clearly that chicken or rabbit tropomyosin could be incorporated by the mutant hearts and that it was sufficient to overcome the factors causing a lack of myofibril formation in the mutant. This finding also suggests that a lack of organized myofibrils is caused primarily by either inadequate levels of tropomyosin or endogenous tropomyosin in mutant hearts is unsuitable for myofibril formation, which we were able to duplicate with the introduction of tropomyosin antibody. Furthermore, incorporation of a specific exogenous protein or antibody into normal and mutant hearts of the Mexican axolotl in whole heart organ culture offers an unique model to evaluate functionalroles of contractile proteins necessary for cardiac development and differentiation.