Jin J P
Department of Medical Biochemistry, University of Calgary Faculty of Medicine, Alberta, Canada.
J Biol Chem. 1995 Mar 24;270(12):6908-16.
Titin (connectin) is a giant protein that forms a single-molecule elastic filament extending from the M-line to the Z-line in the striated muscle sarcomere. The sequence of titin consists mainly of repeats of two types of approximately 100-amino acid motifs (class I and class II that show homology to the fibronectin type III and immunoglobulin-C2 domains, respectively). To investigate the functions of the two classes of titin motifs as the basic units of this large sarcomere organizer molecule, titin cDNA segments encoding single class I or class II or linked class I-II motifs were cloned by polymerase chain reaction from a rat cardiac cDNA library into the T7 RNA polymerase-based pAED4 vector to express non-fusion titin fragments in Escherichia coli. High level expression of the three titin fragments was achieved, and effective rapid purification procedures were developed. The purified titin fragments were verified by their amino acid composition, apparent molecular mass, and charge. Antibodies raised against the genetically expressed titin motifs specifically recognized intact rat cardiac and skeletal muscle titins in Western blotting and immunofluorescence microscopy, confirming the authenticity of the cloned fragments. High beta-sheet contents of these titin motifs indicate a folding state very similar to that of intact native titin. Solid-phase protein-binding assays demonstrated that a single class I motif was able to bind both myosin and F-actin. In comparison, a single class II motif had weaker binding to only F-actin but the fragment containing linked class I and class II motifs showed significantly stronger interactions with both myosin and F-actin. The binding of titin motifs to myosin supports the proposed association of A-band titin with the thick filament, and the novel titin-F-actin interaction was confirmed by F-actin cosedimentation assays, suggesting that titin may also be involved in the structure and/or function of the thin filament.
肌联蛋白（连接蛋白）是一种巨大的蛋白质，它形成一条单分子弹性细丝，从横纹肌肌节的M线延伸至Z线。肌联蛋白的序列主要由两种类型的约100个氨基酸基序的重复序列组成（I类和II类基序分别与纤连蛋白III型结构域和免疫球蛋白C2结构域具有同源性）。为了研究这两类肌联蛋白基序作为这种大型肌节组织者分子基本单元的功能，通过聚合酶链反应从大鼠心脏cDNA文库中克隆编码单个I类或II类或相连的I-II类基序的肌联蛋白cDNA片段，将其插入基于T7 RNA聚合酶的pAED4载体中，以便在大肠杆菌中表达非融合的肌联蛋白片段。实现了这三种肌联蛋白片段的高水平表达，并开发了有效的快速纯化程序。通过氨基酸组成、表观分子量和电荷对纯化的肌联蛋白片段进行了验证。针对基因表达产生的肌联蛋白基序的抗体在蛋白质印迹和免疫荧光显微镜下特异性识别完整的大鼠心脏和骨骼肌肌联蛋白，证实了克隆片段的真实性。这些肌联蛋白基序的高β-折叠含量表明其折叠状态与完整的天然肌联蛋白非常相似。固相蛋白结合试验表明，单个I类基序能够结合肌球蛋白和F-肌动蛋白。相比之下，单个II类基序仅与F-肌动蛋白的结合较弱，但含有相连的I类和II类基序的片段与肌球蛋白和F-肌动蛋白的相互作用明显更强。肌联蛋白基序与肌球蛋白的结合支持了A带肌联蛋白与粗肌丝的假定关联，并且通过F-肌动蛋白共沉降试验证实了新的肌联蛋白-F-肌动蛋白相互作用，这表明肌联蛋白也可能参与细肌丝的结构和/或功能。
