Expression Studies of Other Unconventional Myosins

其他非常规肌球蛋白的表达研究

• 批准号: 10253810
• 负责人: James Sellers
• 金额: $ 68.44万
• 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/10253810
• 关键词: Actins; Back; Behavior; Binding Sites; Biochemical; Biological Assay; Cells; Collaborations; Electrons; Goals; Gold; Head; Head Start Program; Image Analysis; Individual; Kinetics; Label; Measures; Methods; Microfilaments; Molecular; Motor; Movement; Myosin ATPase; Pattern; Positioning Attribute; Proteins; Regulation; Rest; Sampling; Structural Protein; Structure; Tail; Techniques; Universities; base; biophysical techniques; light microscopy; member; microscopic imaging; monomer; optical traps; particle; single molecule

In collaboration with Philipp Kukura of Oxford University, we have used a light microscopy based interferometric scattering technique to examine the processive movement of myosin 5 HMM on actin. By attaching a 20 nm gold particle to the amino-terminus of myosin 5a we are able to measure the movement at sampling rates up to 1000 Hz and follow the movement of the unattached labeled myosin head. Using superresolution methods, we have been able to resolve the single stepping behavior of this myosin into 8 peaks representing individual steps ranging from 11-17 actin monomers with starting head separations of 11, 13, 15, or 17 actin monomers. If the gold bead is placed on the tail of the myosin 5a HMM, we see that the tail transiently moves forward to a position that is further along the actin filament before relaxing back to its rest position. We have begun to analyze the structure and stepping pattern of myosin-6, a processive myosin which moves in the opposite direction on actin compared to other myosins. EM studies show that the angle between the two heads is more variable than in most myosins and that when bound to actin, the motors can be spaced at 13 actins (preferred) or 11 or 15 actin monomers apart. Optical trapping and EM studies demonstrate that the molecule can sometimes take an "inchworm" like step where the two heads occupy closely spaced binding sites. With the lab of Ad Bax we have used NMR to examine the rigidity of the SAH domain of myosin 6.

我们与牛津大学的 Philipp Kukura 合作，使用基于光学显微镜的干涉散射技术来检查肌球蛋白 5 HMM 在肌动蛋白上的持续运动。通过将 20 nm 金颗粒附着到肌球蛋白 5a 的氨基末端，我们能够以高达 1000 Hz 的采样率测量运动，并跟踪未附着的标记肌球蛋白头部的运动。 使用超分辨率方法，我们已经能够将该肌球蛋白的单步行为解析为 8 个峰，代表 11-17 个肌动蛋白单体的各个步骤，起始头部分离为 11、13、15 或 17 个肌动蛋白单体。 如果将金珠放置在肌球蛋白 5a HMM 的尾部，我们会看到尾部短暂地向前移动到沿着肌动蛋白丝更远的位置，然后放松回到其静止位置。 我们已经开始分析肌球蛋白 6 的结构和步进模式，肌球蛋白 6 是一种进行性肌球蛋白，与其他肌球蛋白相比，它在肌动蛋白上的运动方向相反。 EM 研究表明，两个头之间的角度比大多数肌球蛋白的变化更大，并且当与肌动蛋白结合时，马达可以间隔 13 个肌动蛋白（首选）或 11 或 15 个肌动蛋白单体。 光学捕获和电磁研究表明，该分子有时会采取类似“尺蠖”的步骤，其中两个头部占据紧密间隔的结合位点。 在 Ad Bax 实验室中，我们使用 NMR 来检查肌球蛋白 6 SAH 结构域的刚性。