Expression Studies of Other Unconventional Myosins

与牛津大学的Philipp Kukura合作，我们使用了一种基于光学显微镜的干涉散射技术来研究肌球蛋白5 HMM在肌动蛋白上的前进运动。通过在肌球蛋白5a的氨基末端连接一个20 nm的金粒子，我们能够在高达1000赫兹的采样率下测量运动，并跟踪未连接的标记的肌球蛋白头部的运动。使用超分辨方法，我们已经能够将这种肌球蛋白的单步行为分解成8个峰，代表从11-17个肌动蛋白单体到起始头分离11、13、15或17个肌动蛋白单体的各个步骤。如果将金珠放在肌球蛋白5a的尾巴上，我们会看到尾巴暂时向前移动到沿肌动蛋白细丝更远的位置，然后放松到休息位置。我们已经开始分析肌球蛋白-6的结构和步进模式，这是一种前进的肌球蛋白，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，与大多数肌球蛋白相比，两个头部之间的角度变化更大，当与肌动蛋白结合时，马达可以以13个肌动蛋白(首选)或11或15个肌动蛋白单体的间隔排列。光学捕获和EM研究表明，分子有时可以采取类似于尺寸虫的步骤，在那里两个头部占据紧密间隔的结合部位。在Ad Bax的实验室中，我们用核磁共振技术检测了肌球蛋白6的SAH结构域的刚性。

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.