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。使用这项技术，我们能够成像单个的，未标记的肌球蛋白分子5 HMM，以几纳米的精度沿着肌动蛋白移动。该分子的步长为36 nm，移动速度与先前报道的荧光标记肌球蛋白5的移动速度相同。通过将20 nm的金粒子连接到氨基末端，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肌球蛋白头部的运动。有趣的是，即使附着了20纳米的金粒子，肌球蛋白的运动速度也与未标记的分子相同。未标记的头部并不像先前提出的那样自由扩散，而是将大部分时间花在一个固定的、脱离肌动蛋白的位置上，它偶尔会从这个位置探索前向肌动蛋白结合部位。对两个头部的同时跟踪显示，在指南针般的旋转运动中，连续的步伐沿着相同的路径到达肌动蛋白的同一侧，展示了对称的行走模式。对步长的更详细的观察表明，该技术可以准确地检测到在每一步中跨越了多少肌动蛋白单体，这使得我们能够检查头-头间距对步进动力学的影响。在与汤姆·弗里德曼的实验室合作中，我们研究了一种带有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NIH测量了这种蛋白质的ATPase和体外运动性，并与意大利的Marco Capitanio实验室合作，在光学陷阱中测量了这种肌球蛋白的机械性能。我们发现，施加的张力，无论是辅助的还是阻力的，都会降低分子的运行长度。 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。

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.