Regulation and Function of Ligand Binding Sites in Talin

Talin是细胞-基质粘附型连接（粘着斑，FA）的重要组成部分。配体与塔林蛋白结合的动态调节涉及细胞内粘附复合物的机械稳定性和营业额的控制。然而，塔林配体结合位点的调节还没有得到充分的表征。这项拟议的工作将提供一个详细的调查结合位点的活动在C-末端结构域构建的塔林，称为talinC的相互依赖性。talinC的配体结合传递细胞中蛋白质的FA靶向，并允许研究黏着斑蛋白与高亲和力相互作用位点（VBS）的相互作用。在纯化的talinC中，VBS螺旋隐藏在其捆绑结构域中。FA中talinC VBS螺旋的激活被认为涉及整合素和F-肌动蛋白结合。这项工作使用选择性结合位点突变体结合FRAP和FRET为基础的分析荧光标记的蛋白质，以揭示动态协会的talinC变体与和构象重排的脂肪酸。这项工作将提供新的见解，在复杂的配体结合的调节和它的连接到一个假定的机械传感器功能的talinC，此外，有助于在talin的结合位点的调节的分子理解。

Talin is an essential component of cell-matrix adherens type junctions (focal adhesions, FA). Dynamic modulation of ligand binding to talin has been implicated in the control of both the mechanical stability and the turnover of the intracellular adhesion complex. Regulation of ligand binding sites in talin, however, is not characterized sufficiently. This proposed work will provide a detailed investigation of the interdependence of binding site activities in a C-terminal domain construct of talin, termed talinC. Ligand binding of talinC conveys FA targeting of the protein in cells and allows investigation of vinculin interaction with high affinity interaction sites (VBS). In purified talinC, the VBS helices are concealed in its bundled domain structure. Activation of talinC VBS helices in FAs is supposed to involve integrin and F-actin binding. This work uses selective binding site mutants in conjunction with FRAP- and FRET-based analysis of fluorescently labelled protein to reveal the dynamic association of talinC variants with and conformational rearrangements in FAs. The work will provide novel insight in the intricate regulation of ligand binding and its connection to a supposed mechano-sensor function of talinC and, furthermore, contribute to a molecular understanding of the regulation of binding site in talin.