Membrane properties of the OHC system

项目摘要 在这个提议中，我们寻求发展单分子FRET在普雷斯廷。该方法将使我们能够评估 蛋白质的功能使用光学措施。单分子FRET已经在其他蛋白质中开发， 帮助理解其功能。目前，我们还没有一个单一的普雷斯廷的功能测定。 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。 普雷斯廷所属的扩展阴离子转运蛋白家族的成员。现有结构数据 模型数据表明，该家族的成员具有14个跨膜结构域， 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 单体具有门和核心结构域。我们对普雷斯廷进行建模，使其表现得像这些使用电梯的运输机 从收缩状态到扩张状态的机制。这些家庭成员的类似运动 从内部开放到外部开放的构象导致相同的两个部分之间的相对运动 单体的因此，存在门结构域（Tm结构域5-8和12-14）相对于稳定结构域的移动。 含有Tm阴离子结合位点的核心结构域（Tm结构域1-4和9-11）。基于这些模型，我们将 在门控结构域中插入几个半胱氨酸残基，这将允许我们用两个不同的半胱氨酸标记单个二聚体。 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，其显示减少，但 可测量的门控电荷移动（NLC），确认其功能。之间的距离 将荧光团建模为变化30 A。，这将引起FRET效率的显著变化。 建立单分子FRET测定将使我们能够测量生理参数如何影响细胞的生长。 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.