Molecular design of the single-headed processive myosin

单头进行性肌球蛋白的分子设计

• 批准号: 13480215
• 负责人: SUTOH Kazuo
• 金额: $ 9.54万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480215/
• 关键词: Molecular Motor; Processivity; Human myosin; Unconventional Myosin; Recombinant Protein; ミオシン; 滑り運動; プロセッシブ運動; モーティリティーアッセイ

Motor proteins belong one of the classes according to their structure and behavior ; two-headed non-processive, two-headed processive, one-headed non-processive and one-headed processive. Myosin II is in the first class. Myosin V and VI belong to the second class. Myosin I belongs to the third class. One of the kinesin superfamily proteins belong to the last class. In human myosins, myosin IIIA, myosin IIIB, myosin IXA, and myosin IXA are supposed to belong to the last class, although it has not yet been determined experimentally if these myosins are actually one-headed and processive. We have cloned full-length cDNA of human myosin IIIA, IIIB, and IXB from the human brain cDNA library. Cloned myosin IIIA and IXB cDNAs show that there are several types of alternative splicing. We have expressed these cDNAs including several splice variants in Sf9 or high five cells. From the transfected cells, we have purified the expressed proteins for further biochemical and biophysical analyzes. At this stage, we are only partially successful to isolate the proteins due to the formation of inclusion bodies. Various purification procedures have been tried to increase the solubility of expressed proteins. Further studies are required to finally identify the motor characteristics of myosin IIIA, IIIB and IXB.

根据其结构和行为，马达蛋白属于一类：双头非进行性、双头进行性、单头非进行性和单头进行性。肌球蛋白II是一流的。肌球蛋白V和肌球蛋白VI属于第二类。肌球蛋白I属于第三类。运动蛋白超家族中的一种属于最后一类。在人类肌球蛋白中，肌球蛋白IIIA、肌球蛋白IIIB、肌球蛋白IXA和肌球蛋白IXA被认为属于最后一类，尽管还没有实验确定这些肌球蛋白是否真的是单头和进行性的。我们已经从人脑cDNA文库中克隆了人肌球蛋白IIIA、IIIB和IXB的全长cDNA。克隆的肌球蛋白IIIA和IXB cDNA表明存在几种类型的选择性剪接。我们已经在Sf9或High 5细胞中表达了这些cDNA，包括几个剪接变体。从转基因细胞中，我们纯化了表达的蛋白质，用于进一步的生化和生物物理分析。在这个阶段，由于包涵体的形成，我们只成功地分离了部分蛋白质。为了提高表达蛋白质的溶解度，人们尝试了各种纯化方法。需要进一步的研究才能最终确定肌球蛋白IIIA、IIIB和IXB的运动特性。

项目成果

Sakurai, M., Adachi, H., Sutoh, K.: "Mutational analyses of Dictyostelium IQGAP-related protein GApa : possible interaction with small GTPases in cytokinesis"Biosci Biotechnol Biochem.. 65. 1912-1916 (2001)

Sakurai, M., Adachi, H., Sutoh, K.：“盘基网柄菌 IQGAP 相关蛋白 GApa 的突变分析：胞质分裂中与小 GTP 酶的可能相互作用”Biosci Biotechnol Biochem.. 65. 1912-1916 (2001)

Suzuki, Y., Tani, T., Sutoh, K., Kamimura, S.: "Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy"FEBS Letters. 512. 235-239 (2002)

Suzuki, Y.、Tani, T.、Sutoh, K.、Kamimura, S.：“通过基于棱镜的光谱对单个荧光分子的荧光光谱进行成像”FEBS Letters。

Sasaki, N., Ohkura, R., Sutoh, K.: "Dictyostelium myosin II as a model to study the actin-myosin interactions during force generation"J. Muscle Res. Cell Motility. 23(in press). (2003)

Sasaki, N.、Ohkura, R.、Sutoh, K.：“盘基网柄菌肌球蛋白 II 作为研究力生成过程中肌动蛋白-肌球蛋白相互作用的模型”J.

Suzuki Y, Tani T, Sutoh K, Kamimura S: "Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy"FEBS Letters. 512(1-3). 235-239 (2002)

Suzuki Y、Tani T、Sutoh K、Kamimura S：“通过基于棱镜的光谱对单个荧光分子的荧光光谱进行成像”FEBS Letters。

Asukagawa, H., Sutoh, K.: "The alanine-scanning mutagenesis of Dictyostelium myosin II at the ionic interface with actin"Results Probl Cell Differ.. 36. 65-74 (2002)

Asukakawa, H., Sutoh, K.：“盘基网柄菌肌球蛋白 II 在与肌动蛋白的离子界面处的丙氨酸扫描诱变”结果 Probl Cell Differ.. 36. 65-74 (2002)