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首页> 外文期刊>Biochemistry >A recombinant monocysteine mutant (Ser to Cys-155) of fast skeletal troponin T: Identification by cross-linking of a domain involved in a physiologically relevant interaction with troponins C and I
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A recombinant monocysteine mutant (Ser to Cys-155) of fast skeletal troponin T: Identification by cross-linking of a domain involved in a physiologically relevant interaction with troponins C and I

机译：快速骨骼肌钙蛋白T的重组单半胱氨酸突变体（Ser至Cys-155）：通过与涉及肌钙蛋白C和I的生理相关相互作用的结构域交联来鉴定

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摘要

Troponin T (TnT), a subunit of the heterotrimeric troponin (Tn) complex, is essential for the Ca2+ regulation of vertebrate striated muscle contraction both in vivo and in vitro. With the exception of bovine cardiac TnT, all known vertebrate TnT isoforms lack a thiol group, a property which makes the wild-type proteins unsuitable as cross-linking substrate. We generated a mutant human fast skeletal TnT in which Ser(155) was changed to Cys (TnT-Cys(155)). Mutation of this residue in TnT as well as in vitro expression in Escherichia coli and purification of the recombinant mutant protein did not affect its biological properties in terms of in vitro binding to troponin I (TnI), troponin C (TnC), actin-tropomyosin (actin-Tm), and actomyosin ATPase activity. TnT-Cys(155) was labeled with 4-maleimidobenzophenone (BP-TnT(155)) and photo-cross-linked to TnI, TnC, Tm, and all of the thin filament proteins. BP-TnT(155) did not cross-link to Tm and showed weak Ca2+/Mg2+-independent cross-linking with TnI in the binary complex and in the presence of all thin filament protein components. BP-TnT(155) showed Ca2+/Mg2+-dependent cross-linking with TnC in the binary and ternary complexes and Ca2+-favored cross-linking with TnI in the ternary complex. Thus, residue 155 of TnT is within 10 Angstrom (the length of cross-linker) of TnC in the presence or absence of Ca2+ and comes within 10 Angstrom of both TnI and TnC in the presence of Ca2+. TnT residue 155 is in close proximity to or may even partly encompass the Tm binding site. These results suggest that TnT, in association with TnI, may participate in the "information transfer" mediated by the Ca2+ binding signal from TnC to Tm and the region around TnT residue 155 probably acts as a linker between troponin and actin-Tm in this signal transmission process. Our results also suggest that TnT contains at least one Ca2+/Mg2+-dependent TnC binding region located between its Tm and TnI binding regions. A recombinant truncated fragment of TnI, TnI(96-181), containing amino acid residues 96-181 and labeled with BP at Cys-133, failed to cross-link with TnT, indicating that the region around Cys-133 of TnI is not involved in binary interaction with TnT. [References: 50]
机译：肌钙蛋白T（TnT）是异三聚肌钙蛋白（Tn）复合物的一个亚基，对于体内和体外的脊椎动物横纹肌收缩的Ca2 +调节至关重要。除牛心脏TnT外，所有已知的脊椎动物TnT亚型均缺少巯基，该特性使野生型蛋白不适合用作交联底物。我们生成了一个突变的人类快速骨骼TnT，其中Ser（155）变为Cys（TnT-Cys（155））。 TnT中此残基的突变以及在大肠杆菌中的体外表达以及重组突变蛋白的纯化，在体外与肌钙蛋白I（TnI），肌钙蛋白C（TnC），肌动蛋白-原肌球蛋白的结合方面均不影响其生物学特性。 （肌动蛋白-Tm）和肌动球蛋白ATPase活性。 TnT-Cys（155）用4-maleimidobenzophenone（BP-TnT（155））标记，并与TnI，TnC，Tm和所有细丝蛋白进行光交联。 BP-TnT（155）没有与Tm交联，并且在二元复合物中和存在所有细丝蛋白成分的情况下，显示出与TnI弱的不依赖Ca2 + / Mg2 +的交联。 BP-TnT（155）在二元和三元复合物中显示了与TnC的Ca2 + / Mg2 +依赖性交联，在三元复合物中显示了与TnI的Ca2 +有利的交联。因此，在存在或不存在Ca 2+的情况下，TnT的残基155在TnC的10埃之内（交联剂的长度），在存在Ca 2+的情况下在TnI和TnC的10埃之内。 TnT残基155非常接近或什至可以部分包含Tm结合位点。这些结果表明，TnT与TnI相关，可能参与由TnC到Tm的Ca2 +结合信号介导的“信息传递”，并且TnT残基155周围的区域可能充当该信号中肌钙蛋白和肌动蛋白-Tm之间的接头。传输过程。我们的结果还表明，TnT包含至少一个位于其Tm和TnI结合区之间的Ca2 + / Mg2 +依赖性TnC结合区。 TnI的重组截短片段TnI（96-181）含有氨基酸残基96-181，在Cys-133处用BP标记，未能与TnT交联，这表明TnI的Cys-133周围区域不是参与与TnT的二进制交互。 [参考：50]

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