Imaging the structure and dynamics of membrane proteins

膜蛋白的结构和动力学成像

• 批准号: 8344894
• 负责人: Justin Taraska
• 金额: $ 81.68万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344894
• 关键词: Architecture; Behavior; Binding; Biological; Cell membrane; Cells; Complex; Copper; DNA Sequence Rearrangement; Dyes; Environment; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Image; Ions; Life; Light; Maps; Measures; Membrane; Membrane Proteins; Methods; Molecular; Monitor; Movement; Nickel; Post-Translational Protein Processing; Protein Engineering; Proteins; Regulation; Signal Transduction; Structure; Synapses; Transition Elements; Vesicle; Work; extracellular; falls; in vivo; protein complex; protein structure; receptor; tool; trafficking

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.

要求1 荧光共振能量转移（FRET）是一种测量分子距离变化的有力工具，其中供体吸收的光能被转移到附近的受体。 FRET的效率随着两个分子之间距离的六次方而福尔斯，使得FRET对距离的变化非常敏感。 然而，FRET只能在很窄的距离范围内有效地测量距离，这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）作为小荧光供体染料的能量受体，以映射工程蛋白质的构象重排。这些荧光方法比经典的FRET在更短的距离上工作，使用具有更短接头的更小染料，并且对通常与其他方法相关的取向问题不敏感。 目的2 细胞膜蛋白存在于复杂的分子环境中。例如，许多膜蛋白组装为同源或异源复合物。此外，几十个结合伴侣可以瞬时与膜蛋白相互作用，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理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。这些研究将帮助我们了解这些蛋白质是如何结构化的，它们的复合物是如何组装的，以及这些复合物的运输是如何在活细胞内调节的。