Studies Of Myosin V

肌球蛋白 V 的研究

• 批准号: 7158529
• 负责人: JAMES R. SELLERS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7158529
• 关键词: actins; active sites; adenosinetriphosphatase; binding sites; biomechanics; calmodulin; enzyme activity; fluorescence microscopy; fluorimetry; intracellular transport; microfilaments; molecular /cellular imaging; myosins; nanotechnology; protein protein interaction; protein structure; protein structure function; protoplasm motility; structural biology; tissue /cell culture

Recent studies provide strong evidence that single myosin class V molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, 'hand over hand', for each diffusional encounter. The mechanisms regulating myosin-V's processivity remain unknown. Here, we have used an optical-tweezers-based transducer to measure the effect of load on the mechanical interactions between rabbit skeletal F-actin and a single head of mouse brain myosin-V, which produces its working stroke in two phases. We found that the lifetimes of the first phase of the working stroke changed exponentially and about 10-fold over a range of pushing and pulling forces of +/- 1.5 pN. Stiffness measurements suggest that intramolecular forces could approach 3.6 pN when both heads are bound to F-actin, in which case, extrapolation would predict the detachment kinetics of the front head to slow down 50-fold and the kinetics of the rear head to accelerate, respectively. This synchronizing effect on the chemo-mechanical cycles of the heads increases the probability of the trail head detaching first and causes a strong increase in the number of forward steps per diffusional encounter over a system with no strain dependence. Myosin V has an extremely long neck containing six calmodulin-binding IQ motifs that allows it to take multiple 36 nm steps corresponding to the pseudo-repeat of actin. To further investigate how myosin V moves processively on actin filaments, we altered the length of the neck by adding or deleting IQ motifs in myosin constructs lacking the globular tail domain. These myosin V IQ mutants were fluorescently labeled by exchange of a single Cy3-labeled calmodulin into the neck region of one head. We measured the step-size of these individual IQ mutants with nanometer precision and sub-second resolution using FIONA. The step-size was proportional to neck length for constructs containing 2, 4, 6 and 8 IQ motifs, providing strong support for the swinging lever arm model of myosin motility. In addition, the kinetics of stepping provided additional support for the hand-over-hand model whereby the two heads alternately assume the leading position. Interestingly, the 8 IQ myosin V mutants gave a broad distribution of step-sizes with multiple peaks, suggesting that this mutant has many choices of binding sites on an actin filament. These data demonstrate that the step-size of myosin V is affected by the length of its neck and is not solely determined by the pseudo-repeat of the actin filament.

最近的研究提供了强有力的证据表明，单个肌球蛋白V类分子运输囊泡和细胞器procedescent沿着F-肌动蛋白，采取几个36纳米的步骤，“手在手上”，为每个扩散遇到。调节肌球蛋白-V的持续合成能力的机制仍然未知。在这里，我们已经使用了一个光镊为基础的传感器来测量兔骨骼F-肌动蛋白和小鼠脑肌球蛋白-V，产生其工作行程在两个阶段的一个单一的头之间的机械相互作用的负载的影响。我们发现，工作冲程的第一阶段的寿命呈指数变化，在+/- 1.5 pN的推力和拉力范围内变化约10倍。刚度测量表明，当两个头部都与F-肌动蛋白结合时，分子内力可以接近3.6 pN，在这种情况下，外推法将预测前头部的分离动力学减慢50倍，后头部的动力学分别加速。这种同步作用的化学机械循环的头部增加的可能性，尾部头部分离第一，并导致一个强大的增加，在向前的步骤数的系统，没有应变依赖性的每个扩散遇到。 肌球蛋白V有一个非常长的颈部，包含六个钙调蛋白结合IQ基序，使其能够采取多个36 nm的步骤，对应于肌动蛋白的假重复。为了进一步研究肌球蛋白V如何在肌动蛋白丝上向前移动，我们通过在缺乏球状尾结构域的肌球蛋白结构中添加或删除IQ基序来改变颈部的长度。这些肌球蛋白V IQ突变体通过将单个Cy 3标记的钙调蛋白交换到一个头部的颈部区域来荧光标记。我们使用FIONA以纳米精度和亚秒级分辨率测量了这些个体IQ突变体的步长。对于含有2、4、6和8个IQ基序的结构，步长与颈部长度成比例，为肌球蛋白运动的摆动杠杆臂模型提供了强有力的支持。此外，步进的动力学为双手交替模式提供了额外的支持，即两个头部交替占据领先位置。有趣的是，8个IQ肌球蛋白V突变体给出了具有多个峰的宽步长分布，这表明该突变体在肌动蛋白丝上具有许多结合位点的选择。这些数据表明，肌球蛋白V的步长的影响，其颈部的长度，而不仅仅是由假重复的肌动蛋白丝。