Expression Studies of Other Unconventional Myosins

与牛津大学的Philipp Kukura合作，我们使用了基于光学显微镜的干涉散射技术来研究肌球蛋白5 HMM在肌动蛋白上的进行性运动。通过将20 nm的金颗粒连接到肌球蛋白5a的氨基末端，我们能够以高达1000 Hz的采样率测量运动，并跟踪未连接的标记肌球蛋白头部的运动。 使用超分辨方法，我们已经能够解决这个肌球蛋白的单步行为为8个峰，代表从11-17个肌动蛋白单体的起始头分离的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 HMM的尾部，我们可以看到，在放松回到其静止位置之前，尾部会短暂地向前移动到沿着肌动蛋白丝更远的位置。 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，它在肌动蛋白上以相反的方向移动。 EM研究表明，两个头部之间的角度比大多数肌球蛋白更可变，并且当与肌动蛋白结合时，马达可以间隔13个肌动蛋白（优选）或11或15个肌动蛋白单体。 光学捕获和EM研究表明，分子有时可以采取“尺蠖”般的步骤，其中两个头部占据紧密间隔的结合位点。 在Ad Bax实验室，我们使用NMR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.