Expression Studies of Other Unconventional Myosins

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。 肌球蛋白7a是一种基于肌动蛋白的运动蛋白，对视觉和听觉至关重要。肌球蛋白7a的突变导致Usher综合征1型，这是人类最常见和最严重的聋盲形式。控制其机械化学的分子机制仍然知之甚少，主要是由于难以纯化稳定，完整的蛋白质。在这里，我们重组生产完整的人类肌球蛋白7a全酶在昆虫细胞和其生化和运动特性的特点。与主要与钙调蛋白相关的果蝇直系同源物不同，我们发现人肌球蛋白7a利用了包括调节轻链、钙调蛋白和钙调蛋白样蛋白4（CALML 4）在内的轻链的独特组合。我们的研究结果进一步表明，CALML 4不作为Ca 2+传感器，但在维持杠杆臂结构功能完整性方面起着至关重要的作用。利用我们的重组蛋白系统，我们纯化了两个肌球蛋白7a剪接异构体，这两个异构体已被证明是差异表达的沿着耳蜗tonotopic轴。我们表明，他们具有不同的机械酶的特性，尽管只有11个氨基酸在其N端不同。使用单分子在体外运动分析，我们证明，人肌球蛋白7a作为一个自抑制单体存在，并可以移动procancer沿着肌动蛋白时，人工二聚化或绑定货物衔接蛋白，如MyRIP。这些结果表明，肌球蛋白7a可以在感觉系统中发挥多种作用，如转运蛋白或锚/力传感器。此外，我们的研究强调，人类肌球蛋白7a已经进化出独特的调节元件，使其机械性能的精确调谐适合哺乳动物的听觉功能。 在与约翰·哈默的合作中，我们已经开始研究肌球蛋白19，一种线粒体相关的肌球蛋白，以补充正在进行的细胞生物学研究。

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. Myosin 7a is an actin based motor protein essential for vision and hearing. Mutations of myosin 7a cause Usher syndrome type 1, the most common and severe form of deaf blindness in humans. The molecular mechanisms that governs its mechanochemistry remain poorly understood, primarily due to the difficulty of purifying stable, intact protein. Here, we recombinantly produce the complete human myosin 7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog which primarily associates with calmodulin, we found that human myosin 7a utilizes a unique combination of light chains including regulatory light chain, calmodulin, and calmodulin like protein 4 (CALML4). Our results further reveal that CALML4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arms structural functional integrity. Using our recombinant protein system, we purified two myosin 7a splicing isoforms which have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechano enzymatic properties despite differing by only 11 amino acids at their N termini. Using single molecule in vitro motility assays, we demonstrate that human myosin 7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins such as MyRIP. These results suggest that myosin 7a can serve multiple roles in the sensory systems such as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin 7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions. In collaboration with John Hammer we have begun to study myosin 19 a mitochondrial associated myosin to complement ongoing cell biological studies.