The Effect Of Deafness Associated Mutations on MyosinVIIA Function

描述(申请人提供)：肌球蛋白VIIA是一种细胞运动蛋白，在多种组织中表达，包括内耳和视网膜。肌球蛋白VIIA基因突变与多种感觉神经元疾病有关，包括以耳聋、前庭功能障碍和失明(视网膜色素变性)为特征的Usher综合征1B，人类的两种常染色体隐性听力障碍DFNA11和DFNB2，以及小鼠的Shaker-1表型。尽管肌球蛋白VIIA基因在这些综合征的病因学中很重要，但关于这种马达蛋白的分子机制尚缺乏信息。本研究的目的是通过研究肌球蛋白VIIA突变对ATPase活性和运动性的影响，确定肌球蛋白VIIA是否是一种进行性运动，从而表征肌球蛋白VIIA蛋白的运动特性。肌球蛋白VIIA的野生型和三个摇床突变体的截短形式将与钙调蛋白轻链一起在杆状病毒/昆虫表达系统中表达。我们将进行稳态实验，以确定马达的总水解率或“输出”，并进行瞬时动力学测量，以确定底物与野生型和突变型肌球蛋白VIIA和肌动肌球蛋白VIIA复合体结合和产物解离的选定速率常数。因此，肌球蛋白VIIA ATPase周期的限速步骤和马达的占空比(肌球蛋白与肌动蛋白紧密结合的ATPase周期时间的部分)将被确定。由这些研究确定的速率常数将有助于我们设计分析性的超速离心和体外运动研究。将进行分析性超速离心实验，以便准确测量野生型和突变型肌球蛋白VIIA蛋白的分子量。这些研究还将确定肌球蛋白VIIA是否形成二聚体。野生型和突变型肌球蛋白VIIA的速度将通过测量罗丹明标记的肌动蛋白细丝在肌球蛋白VIIA分子上滑动的运动性(体外运动性)来确定。在单分子运动性测量中，将荧光标记的CaM轻链添加到肌球蛋白VIIA上，用全内反射荧光显微镜(TIRF)测量分子的运动。游程长度(肌球蛋白VIIA分子在肌动蛋白细丝上移动而不与之解离的距离)，步长(两个头之间的距离)，从而决定不同肌球蛋白VIIA结构进行的程度。这些研究将有助于阐明肌球蛋白VIIA马达蛋白的分子机制，加深我们对肌球蛋白VIIA相关的耳聋和失明的认识。我们将对野生型和突变型肌球蛋白VIIA蛋白的生化、结构和运动特性进行研究，以阐明肌球蛋白VIIA相关耳聋、前庭功能障碍和失明的分子机制。

DESCRIPTION (provided by applicant): Myosin VIIA is a cellular motor protein which is expressed in a several tissues including the inner ear and the retina. Mutations in the myosin VIIA gene are associated with a variety of sensory neuronal disorders including the Usher syndrome 1B, characterized by deafness, vestibular dysfunction and blindness (retinitis pigmentosa), and two autosomal recessive hearing disorders, DFNA11 and DFNB2 in humans, and the shaker-1 phenotype in mice. Despite the importance of the myosin VIIA gene in the etiology of these syndromes, there is a lack of information on the molecular mechanism of this motor protein. The goal of this proposal is to characterize the motor properties of the myosin VIIA protein by determining if myosin VIIA is a processive motor and by studying the effect of the myosin VIIA mutations on the ATPase activity and motility. Truncated forms of the wild type and three shaker mutants of the myosin VIIA will be expressed in a baculovirus/insect expression system along with the calmodulin light chain. We will carry out steady-state experiments to determine the overall rate of the hydrolysis or the "output" of the motor, and transient kinetic measurements to determine selected rate constants for the substrate binding to and product dissociation from the wild type and mutant myosin VIIA and actomyosin VIIA complexes. Thus the rate limiting step of the myosin VIIA ATPase cycle and the duty ratio (the fraction of the ATPase cycle time the myosin spends tightly bound to actin) of the motor will be determined. The rate constants determined by these studies will aid us in designing the analytical ultracentrifugation and the in vitro motility studies. Analytical ultracentrifugation experiments will be conducted so that the molecular weight of the wild type and mutant myosin VIIA proteins can be accurately measured. These studies will also determine whether myosin VIIA forms dimers. The velocity of the wild type and mutant myosin VIIA will be determined by measuring the motility of the rhodamin labeled actin filaments sliding over the myosin VIIA molecules (in vitro motility). In the single molecule motility measurements, fluorescently labeled CaM light chain will be added to the myosin VIIA and the movement of the molecule will be measured by Total Internal Reflection Fluorescence microscopy (TIRF). The run length (the distance that the myosin VIIA molecule moves on the actin filament without dissociating from it), the step size (the distance between the two heads) thus the extent to which the different myosin VIIA constructs are processive will be determined. These studies will aid to elucidate the molecular mechanism of the myosin VIIA motor protein and increase our understanding of the myosin VIIA related deafness, and blindness. The biochemical, structural and motile properties of the wild type and mutant myosin VIIA proteins will be studied, to delineate the molecular mechanism of the myosin VIIA related deafness, vestibular dysfunction and blindness.