De Lozanne A, Berlot C H, Leinwand L A, Spudich J A
Department of Cell Biology, Stanford University School of Medicine, California 94305.
J Cell Biol. 1987 Dec;105(6 Pt 2):2999-3005. doi: 10.1083/jcb.105.6.2999.
The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known. Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation. We report here the expression in Escherichia coli of a 1.5-kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail. The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E. coli extracts. The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase. Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.
肌球蛋白尾部的氨基酸序列决定了肌球蛋白分子组装到肌球蛋白丝中的具体方式，但分子间相互作用的细节尚不清楚。基因工程改造的肌球蛋白尾部片段的表达将有助于研究对肌球蛋白丝形成及其调控重要的序列。我们在此报告，在大肠杆菌中表达了盘基网柄菌肌球蛋白重链基因的一个1.5 kb片段，该片段编码肌球蛋白尾部的一个58-kD片段。表达的蛋白（DdLMM-58）从大肠杆菌提取物的可溶部分纯化至均一。通过以下标准发现表达的蛋白具有功能：（a）在电子显微镜下它呈现为一条74 nm长的杆状，这是500个氨基酸的α-螺旋卷曲螺旋的预测长度；（b）它组装成丝状结构，显示出在肌肉肌球蛋白天然丝中发现的典型的14 nm轴向周期性；（c）其组装成丝的过程显示出与盘基网柄菌肌球蛋白相同的离子强度依赖性；（d）它作为盘基网柄菌肌球蛋白重链激酶的底物，该激酶在趋化信号作用下使肌球蛋白磷酸化；（e）其磷酸化形式具有与磷酸化的盘基网柄菌肌球蛋白重链相同的磷酸氨基酸和相似的磷酸肽图谱；（f）它与肌球蛋白竞争重链激酶。因此，丝形成和磷酸化所需的所有信息都包含在这个表达的蛋白中。
