Expression Studies of Other Unconventional Myosins

与牛津大学的Philipp Kukura合作，我们使用基于光学显微镜的干涉散射技术来检查肌动蛋白上肌球蛋白5 HMM的过程运动。利用这项技术，我们能够以几纳米的精度成像单个未标记的肌凝蛋白5 HMM分子沿着肌动蛋白移动。分子以36纳米的速度移动，与先前报道的荧光标记肌球蛋白5相同。通过将20 nm的金颗粒连接到氨基端，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。有趣的是，即使有一个20纳米的金颗粒附着，肌凝蛋白的移动速度也与未标记的分子相同。未标记的头部并不像之前提出的那样自由扩散，而是大部分时间都处于固定的，远离肌动蛋白的位置，偶尔从那里探索肌动蛋白的结合位点。同时对两个头部的跟踪显示，连续的脚步沿着相同的路径走到肌动蛋白的同一侧，像指南针一样旋转，显示出对称的行走模式。对步长更详细的观察表明，该技术可以精确地检测到在每一步中跨越了多少肌动蛋白单体，这使我们能够检查头-头间距对步长动力学的影响。我们与Tom Friedman的实验室合作，研究了一种携带耳聋相关突变的肌球蛋白-15的分子特性。我们已经表达和纯化了肌球蛋白5b，并在美国国立卫生研究院测量了atp酶和这种蛋白质的体外运动性，并与意大利的马可·卡皮塔尼奥实验室合作，在光学陷阱中测量了这种肌球蛋白的机械特性。我们表明，施加的张力，无论是辅助的还是电阻的，都会降低分子的运行长度。

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. Using this technique we were able to image single, unlabeled molecules of myosin 5 HMM move along actin with a precision of a few nanometers. The molecule took 36 nm steps and moved at the same speed as previously reported for fluorescently-labeled myosin 5. By attaching a 20 nm gold particle to the amino-terminus we are able to measure the movement at sampling rates up to 1000 Hz and follow the movement of the unattached labeled myosin head. Interesting, even with a 20 nm gold particle attached the myosin moves at the same velocity as the unlabeled molecule. The unlabeled head does not freely diffuse as previously proposed, but rather spends most of its time in a fixed, off actin, position from which it occasionaly explores the forward actin binding sites. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. More detailed observations of the step sizes reveals that the technique can precisely detect how many actin monomers are spanned during each step which allows us to examine the effect of head-head spacing on the stepping kinetics. In collaboration with the lab of Tom Friedman we have examined the molecular properties of a myosin-15 bearing a deafness associated mutation. We have expressed and purified myosin-5B and have measured the ATPase and in vitro motility of this protein at NIH and have collaborated with Marco Capitanio's lab in Italy measuring the mechanical properties of this myosin in an optical trap. We show that imposed strain, either assisting or resistive lowers the run length of the molecule. 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.