Imaging the structure and dynamics of membrane proteins

目标1 荧光共振能量转移(FRET)是一种测量分子距离变化的强有力的工具，它是将供体吸收的光能转移到附近的受体。FRET的效率随着两个分子之间距离的6次方而下降，这使得FRET对距离的变化非常敏感。然而，FRET只能在很小的距离范围内有效地测量距离，这些距离并不总是很适合研究蛋白质的分子内运动。我们正在开发快速高通量的方法，使用过渡金属离子(镍和铜)作为小型荧光供体染料的能量受体，以绘制工程蛋白的构象重排图。这些荧光方法比传统的FRET方法工作距离更短，使用的染料更小，连接子更短，而且对通常与其他方法相关的取向问题不那么敏感。 目标2 细胞膜蛋白存在于复杂的分子环境中。例如，许多膜蛋白以同聚体或异构体复合体的形式组装。此外，数十个结合伙伴可能会瞬间与膜蛋白相互作用，以调节它们的行为或运输。最后，蛋白质的结构受到翻译后修饰和天然膜环境的影响。了解这些复杂的结构参数对于了解膜蛋白的功能和调节是必要的。我们正在使用FRET来绘制天然生物膜内膜蛋白的结构图。这些研究将帮助我们了解这些蛋白质是如何结构的，它们的复合体是如何组装的，以及这些复合体在活细胞内的运输是如何调节的。

Aim 1 Fluorescence resonance energy transfer (FRET), in which light energy absorbed by a donor is transferred to a nearby acceptor, is a powerful tool for measuring changes in molecular distances. The efficiency of FRET falls off with the sixth power of the distance between the two molecules, making FRET very sensitive to changes in distance. However, FRET can measure distances effectively only in a narrow range of distances that are not always well suited to study intramolecular movements in proteins. We are developing rapid high throughput methods that use transition metal ions (nickel and copper) as energy acceptors for small fluorescent donor dyes to map the conformational rearrangements of engineered proteins. These fluorescent methods work over shorter distances than classical FRET, use smaller dyes with shorter linkers, and are not as sensitive to the orientation problems usually associated with other methods. Aim 2 Membrane proteins in cells exist in a complex molecular environment. For example, many membrane proteins assemble as homomeric or heteromeric complexes. Furthermore, dozens of binding partners may transiently interact with membrane proteins to modulate their behavior or trafficking. Finally, the architecture of a protein is influenced by post-translational modifications and the native membrane environment. Understanding these complex structural parameters is necessary for understanding the function and regulation of membrane proteins. We are using FRET to map the structures of membrane proteins within native biological membranes. These studies will help us understand how these proteins are structured, how their complexes assemble, and how the trafficking of these complexes is regulated within living cells.