Studies Of Myosin V

肌球蛋白 V 的研究

• 批准号: 8344781
• 负责人: James Sellers
• 金额: $ 11.95万
• 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/8344781
• 关键词: ATP phosphohydrolase; Actins; Adopted; Affect; Atomic Force Microscopy; Binding; Biological Assay; Calcium; Complement; Cryoelectron Microscopy; F-Actin; Genes; Goals; Hand; Head; Image; Image Analysis; In Vitro; Mammals; Mechanics; Microscopic; Molecular; Motor; Mutate; Myosin ATPase; Myosin Type V; Neck; Negative Staining; Organelles; Positioning Attribute; Property; Regulation; SWI1; Speed; Structure; Techniques; Total Internal Reflection Fluorescent; Transport Vesicles; Walking; arm; cell motility; mutant; optical traps; particle; single molecule

There are three myosin V genes in mammals, termed Va, Vb and Vc. Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have used negative staining and cryo-electron microscopy to examine the structure of myosin V that is walking on actin. These images give clear pictures of myosin V molecules with both heads attached to actin and will allow us to make observations about lever arm position and stiffness. We are examining the structure of actin-bound myosin V mutants in which the spacing between IQ motifs were altered and will complement these studies with in vitro motility assays and optical trapping. We also examined the structure of mutant myosin V molecules in which the neck region was altered by changing the number of IQ motilfs. These studies demonstrate that the neck of myosin V has evolved to allow it to take 36 nm strides along actin which matches the helical repeat. We have mutated the switch-1 region of myosin V (S217A) and found that this mutant alters the duty ratio and effects processivity of the motor. Switch-2 mutants affect the speed of translocation and the ADP release rate. Optical trapping studies show that under certain levels of strain, the powerstroke of myosin V attached to actin can be reversed which may aid in maintaining myosin attachment during stall. We have begun to study the mechanical properties of myosin Vc and the regulatory properties of myosin Vb.

在哺乳动物中存在三种肌球蛋白V基因，称为Va、Vb和Vc。 最近的研究提供了强有力的证据表明，单一的V类肌球蛋白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，为每个扩散遇到。 我们证明了肌球蛋白的ATP酶活性需要钙才能达到最大活性，并表明在没有钙的情况下，肌球蛋白V采用折叠的无活性结构。我们使用负染色和冷冻电子显微镜来检查在肌动蛋白上行走的肌球蛋白V的结构。 这些图像给出了清晰的肌球蛋白V分子的图像，其两个头部都连接到肌动蛋白上，并将使我们能够观察杠杆臂的位置和刚度。我们正在研究肌动蛋白结合的肌球蛋白V突变体的结构，其中IQ基序之间的间距被改变，并将补充这些研究与体外运动分析和光捕获。 我们还研究了突变型肌球蛋白V分子的结构，其中颈部区域通过改变IQ motilfs的数量而改变。 这些研究表明，肌球蛋白V的颈部已经进化到允许它沿着与螺旋重复序列相匹配的肌动蛋白沿着移动36 nm。 我们突变了肌球蛋白V（S217 A）的开关-1区域，发现该突变体改变了占空比并影响马达的持续合成能力。 开关-2突变体影响易位速度和ADP释放速率。 光学捕获研究表明，在一定程度的应变，附着到肌动蛋白的肌球蛋白V的powerstroke可以逆转，这可能有助于在失速期间保持肌球蛋白附着。 我们已经开始研究肌球蛋白Vc的机械性质和肌球蛋白Vb的调节性质。