Membrane properties of the OHC system

项目摘要 在这项建议中，我们寻求在Prestin中开发单分子FRET。该方法将使我们能够评估 用光学方法测量蛋白质的功能。单分子FRET已经在其他蛋白质中被开发出来 有助于了解它们的功能。目前，我们还没有一个单一的功能分析的Prestin 分子分辨率。 实验的基本原理是基于从几个结构中插入的结构数据 Prestin所属的扩展阴离子转运蛋白家族的成员。可用的结构数据在一起 通过模拟数据表明，该家族成员有14个跨膜结构域，其中 对每个单体重复7-7结构。这些蛋白质的功能单位似乎是二聚体。每个人 单体有一个门和核心区。我们模拟Prestin的行为，就像这些使用电梯的运输者 从收缩状态转换到扩展状态的机制。在这些家庭成员中有类似的运动 从内部开放到外部开放的构象导致同一构象的两个部分之间的相对运动 单体。因此，存在栅极区域(TM区域5-8和12-14)相对于稳定器的移动 包含TM阴离子结合部位的核心区(TM区1-4和9-11)。基于这些模型，我们将 在GATE结构域中插入几个半胱氨酸残基，使我们能够用两个不同的二聚体标记单个二聚体 具有FRET活性的荧光团。我们已经产生了一种不含半胱氨酸的蛋白质，它显示出减少但 可测量的门控电荷移动(NLC)，确认其功能。两者之间的距离 荧光团被模拟为变化30A0，这将引起FRET效率的显著变化。 建立单分子FRET分析将使我们能够测量生理参数如何影响 蛋白质的功能。

Project Summary In this proposal we seek to develop single molecule FRET in prestin. The method will allow us to assess the function of the protein using optical measures. Single molecule FRET has been developed in other proteins to aid in the understanding of their function. Presently, we do not have a functional assay of prestin with a single molecule resolution. The rationale for the experiments is based on structural data interpolated from the structure of several members of the extended anion transporter family to which prestin belongs. Available structural data together with modeling data suggest that members of this family have 14 transmembrane domains with an inverted repeat 7+7 structure to each monomer. The functional units of these proteins appear to be dimers. Each monomer has a gate and core domain. We model prestin to behave like these transporters using an elevator mechanism to move from contracted to expanded states. An analogous movement in these family members from inside open to outside open conformations result in a relative motion between two parts of the same monomer. Thus, there is a movement of the gate domain (Tm domains 5-8 and 12-14) relative to the stable core domain (Tm domains 1-4 and 9-11) containing the Tm anion binding site. Based on these models we will insert several cysteine residues in the gate domain that will allow us to label a single dimer with two different fluorophores with FRET activity. We have already generated a cysteine free protein that shows reduced but measurable gating charge movement (NLC), confirming its functionality. The distance between the fluorophores is modelled to vary by 30A0 that would give rise to a significant change in FRET efficiency. Establishing a single molecule FRET assay will allow us to measure how physiological parameters affect the function of the protein.