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| Author(s): |
Bettina Malnic
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 1995-08-01 |
| Examining board members: |
Fernando de Castro Reinach;
Paulo Lee Ho;
Roy Edward Larson;
Glaucius Oliva;
Sérgio Verjovski Almeida
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| Advisor: | Fernando de Castro Reinach |
| Abstract | |
The contraction of skeletal muscle is regulated by troponin and tropomyosin in a Ca2+ dependent manner. The troponin complex consists of three subunits: troponin C (TnC), troponin I (TnI) and troponin T (TnT). Troponin C is the Ca2+ binding subunit, TnI is the inhibitory subunit and TnT binds tightly to tropomyosin. TnI and TnT are highly insoluble proteins at low ionic strengths, unless they are complexed with TnC. The troponin complex can be reconstituted \"in vitro\" from the isolated subunits simply by mixing the subunits at equimolar ratios in urea, which is then removed by dialysis. In the first part of this work a vector for the co-expression of TnC, TnI and TnT in E.coli was constructed. Using this vector we were able to produce a functional troponin complex assembled \"in vivo\" in the E.coli cytoplasm The presence of TnT is required for the Ca2+ dependente regulation of the skeletal muscle contraction. The role of TnT in conferring full Ca2+ sensitivity to the ATPase activity of acto-myosin was analyzed. Deletion mutants of TnT were constructed by site-directed mutagenesis and expressed in E.coli. Troponin complexes containing the TnT deletion mutants and/or TnI deletion mutants, were reconstituted and analyzed in thin filament binding assays and in ATPase activity assays. Based on these studies, TnT was subdivided into three domains: the activation domain (comprised of aminoacids 1-157), the inhibitory domain (comprised of amino acids 157-216) and the TnC/TnI dimer anchoring domain (aminoacids 216-263). We demonstrated that the TnC/TnI is anchored to the thin filament through interaction between the amino-terminal domain of TnI and the region comprised of aminoacids 216-263 of TnT. A model for the role of TnT in the Ca2+ dependent regulation of muscle contraction is proposed. (AU) | |