Force Dependent Unbinding of Mammalian Inner Ear Tip Link Proteins

项目总结/摘要 尖端连接是脊椎动物毛细胞中转导复合体的关键组成部分， 将前庭和听觉刺激转化为电信号。尖端连接由以下平行二聚体组成： PCDH 15和CDH 23，通过独特的界面在其N-末端键合，其结构为 最近通过X射线晶体学解决了。然而，毛细胞尖端的生物物理学知之甚少 以及它对机械力的反应。在这个项目中，我们将确定生物物理特性 为了了解耳聋的后果， 发生在键界面的突变。我们将首先合成含有前两个EC的融合蛋白， 域的PCDH 15和CDH 23，并使用动态力谱来描述一个单一的强度 在存在和不存在钙的情况下。接下来，我们将生成一系列双链尖端连接， 通过抗体Fc结构域人工二聚化的蛋白质，并测量它们在力和 横向约束对其动力学的影响。这将回答为什么提示链接已经演变为 双链，以及尖端链接如何能够忠实地识别机械刺激，但仍然能够 在破坏性的声音水平下解开。最后，我们将产生含有已知耳聋的融合蛋白， 这些突变被认为会损害键的完整性。然后我们将测试这些结果 耳聋突变的动态力谱。尖端生物物理特性的阐明 力下的连接键将告知为什么尖端连接已经进化为双链，如何转导 设备能够存活数天或数周，以及如何治疗某些类型的耳聋突变 在未来

Project Summary/Abstract The tip link is a critical component of the transduction complex in vertebrate hair cells, which converts vestibular and auditory stimuli into an electrical signal. The tip link is composed of parallel dimers of PCDH15 and CDH23, bonded at their N-termini through a unique interface, the structure of which was recently resolved by X-ray crystallography. However, little is known about the biophysics of the hair cell tip link and how it responds to mechanical force. In this project, we will determine the biophysical characteristics of the tip link bond both statically and under force in order to understand the consequences of deafness mutations that occur at the bond interface. We will first synthesize fusion proteins containing the first two EC domains of PCDH15 and CDH23, and use dynamic force spectroscopy to describe the strength of a single bond, both in the presence and absence of calcium. Next, we will generate a series of double stranded tip link proteins, artificially dimerized by an antibody Fc domain, and measure both their strength under force and the consequence of lateral constraint on their kinetics. This will answer why the tip link has evolved to be double stranded, and how the tip link is able to faithfully transduce mechanical stimuli, yet still able to unbind at damaging sound levels. Finally, we will generate fusion proteins containing known deafness mutations which are thought to compromise bond integrity. We will then test the consequence of these deafness mutations by dynamic force spectroscopy. Elucidation of the biophysical characteristics of the tip link bond under force will inform why the tip link has evolved to be double stranded, how the transduction apparatus is able to survive for days or weeks, and how certain types of deafness mutations might be treated in the future.