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.

哺乳动物中有三个肌球蛋白V基因，称为VA，VB和VC。 最近的研究提供了有力的证据，表明单级V肌球蛋白分子沿F-肌动蛋白进行过程中运输囊泡和细胞器，并在每次扩散遭遇中采取了几个36 nm的步骤，手工握手。 我们证明了肌球蛋白的ATPase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采用了折叠的，无活性的结构。我们已经使用了阴性染色和冷冻电子显微镜检查在肌动蛋白上行走的肌球蛋白V的结构。 这些图像给出了肌球蛋白V分子的清晰图像，并附有两个头部附着在肌动蛋白上，并且可以使我们对杠杆臂的位置和刚度进行观察。我们正在研究肌动蛋白结合的肌球蛋白V突变体的结构，其中改变了智商基序之间的间距，并将通过体外运动测定和光学诱捕来补充这些研究。 我们还检查了突变肌球蛋白V分子的结构，其中通过改变智商的数量来改变颈部区域。 这些研究表明，肌球蛋白V的颈部已经演变，使其可以沿肌动蛋白进行36 nm的步幅，与螺旋重复相匹配。 我们已经突变了肌球蛋白V（S217A）的开关1区域，发现该突变体会改变电动机的占空比和影响。 开关2突变体会影响易位速度和ADP释放速度。 光学诱捕研究表明，在某些水平的菌株中，肌动蛋白上附着的肌球蛋白V的动力可以逆转，这可能有助于在失速过程中维持肌球蛋白的附着。 我们已经开始研究肌球蛋白VC的机械性能和肌球蛋白VB的调节性能。