EXPRESSION OF MUTANT VERTEBRATE MYOSIN I'S

突变脊椎动物肌球蛋白 I 的表达

• 批准号: 3757680
• 负责人: F WANG
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3757680
• 关键词: Acanthamoeba; Baculoviridae; adenosinetriphosphatase; alternatives to animals in research; brush border membrane; chickens; eukaryote; gene expression; magnesium; mutant; myosins; phosphorylation; serine; site directed mutagenesis

Myosin I's are widely expressed in different tissues and across broad phylogenetic backgrounds. In intestinal epithelial brush borders, myosin I is localized in the microvillus where it bridges gaps between the membrane and the actin bundles. Myosin I is also localized near the actively ruffling edges of migrating cells. We are using the Baculovirus/Sf9 system to express full-length chicken brush border myosin I heavy chain (BBMI HC) along with calmodulin (CaM), and a truncated head fragment. Vertebrate myosin I's are constitutively active whereas myosin I's from low eukaryotes such as Acanthamoeba and Dictyostelium require phosphorylation at a serine located in the myosin head domain for activity. Sequence alignments of vertebrate and Acanthamoeba myosin I's reveal that the vertebrate proteins have a negatively charged amino acid at the position where the Acanthamoeba protein has the phosphorylatable serine. We intend to explore, using site-directed mutagenesis and the Baculovirus/Sf9 system, whether a negative charge at this site is essential for actin-activated MgATPase activity and in vitro motility.

肌球蛋白I广泛表达于不同的组织中， 系统发育背景 在肠上皮刷状缘，肌球蛋白 I定位于微绒毛中，在微绒毛中，I桥接微绒毛之间的间隙。 膜和肌动蛋白束。 肌球蛋白I也定位于 活跃地弄皱迁移细胞的边缘。 我们使用 杆状病毒/Sf 9系统表达全长鸡刷状缘肌球蛋白 I重链（BBMI HC）沿着与钙调蛋白（CaM），以及截短的头部 碎片 脊椎动物肌球蛋白I具有组成性活性， I来自低等真核生物如阿米巴和网骨藻 位于肌球蛋白头部结构域的丝氨酸磷酸化， 活动 脊椎动物和阿米巴肌球蛋白I的序列比对 揭示了脊椎动物蛋白质具有带负电荷的氨基酸 在阿米巴蛋白具有可磷酸化的 丝氨酸 我们打算利用定点突变和 杆状病毒/Sf 9系统，该位点的负电荷是否 对肌动蛋白激活的MgATPase活性和体外运动性至关重要。