Imaging the structure and dynamics of membrane proteins

膜蛋白的结构和动力学成像

• 批准号: 8746663
• 负责人: Justin Taraska
• 金额: $ 32.87万
• 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/8746663
• 关键词: Adopted; Behavior; Binding; Biological; Cells; Cobalt; Complex; Copper; DNA Sequence Rearrangement; Data; Dyes; Environment; Fluorescein; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Image; In Vitro; Ions; Life; Ligand Binding; Ligands; Light; Maleimides; Maps; Measurement; Measures; Membrane; Membrane Proteins; Metals; Methods; Modeling; Molecular; Molecular Conformation; Motion; Movement; Nickel; Protein Dynamics; Protein Engineering; Proteins; Regulation; Signal Transduction; Structural Models; Structure; Synapses; System; Testing; Transition Elements; Vesicle; Work; Zinc; bimanes; extracellular; falls; in vivo; maltose-binding protein; molecular dynamics; protein structure; receptor; simulation; system architecture; tool

Aim 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 intra-molecular 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 (bimane) to map the conformational rearrangements of engineered proteins. These transition metal ion FRET (tmFRET) 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. In this work, we have used tmFRET to map 10 unique distances in the model protein Maltose Binding Protein (MBP) in both the ligand-bound (HOLO) and ligand-free (APO) state. We have mapped distances between two donor dyes (monobromo-bimane and fluorescein-5-maleimide) and the two acceptor metals (nickel and copper). This has given us a total of 40 independent distance measurements in MBP. When these distances were compared to the x-ray crystal structure of MBP our tmFRET distances match the x-ray crystal structure to within a few angstroms. Furthermore, tmFRET was able to accurately detect structural changes in the protein during ligand binding. With the above experimental data, we next tested if distances derived with tmFRET could be used to guide molecular dynamics simulations. In these tmFRET-constrained simulations, MBP was allowed to move from the ligand-bound state to the APO state. Without tmFRET-derived distance constraints, the simulations did find the APO conformation of MBP. Simulations that contained the tmFRET-derived distances rapidly adopted the APO state. We conclude that tmFRET can be used to drive the conformational folding of proteins structures to an accuracy of a few angstroms. We have continued to develop this method as applied to other proteins. We have explored new metals including zinc and cobalt for binding and fluorescence effects in these systems. We have also developed new structural models of the angstrom-scale architecture of these systems and how they can be used to map other proteins in vitro or in vivo. These systems have been tested in living cells. Thus, tmFRET can be applied to proteins in a living cell environment to map the conformational dynamics of proteins in vivo at the angstrom scale.

目的 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分子内的运动。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（bimane）的能量受体，以映射工程蛋白质的构象重排。这些过渡金属离子FRET（tmFRET）荧光方法比经典FRET在更短的距离上工作，使用具有更短连接体的更小染料，并且对通常与其他方法相关的取向问题不敏感。 在这项工作中，我们使用tmFRET映射10个独特的距离在模型蛋白麦芽糖结合蛋白（MBP）在配体结合（HOLO）和配体自由（APO）状态。我们绘制了两个供体染料（monobromo-bimane和荧光素-5-马来酰亚胺）和两个受体金属（镍和铜）之间的距离。这给了我们总共40个独立的距离测量MBP。当将这些距离与MBP的X射线晶体结构进行比较时，我们的tmFRET距离与X射线晶体结构在几埃内匹配。此外，tmFRET能够准确地检测配体结合过程中蛋白质的结构变化。 利用上述实验数据，我们接下来测试了用tmFRET导出的距离是否可以用于指导分子动力学模拟。在这些tmFRET约束的模拟中，允许MBP从配体结合状态移动到APO状态。在没有tmFRET导出的距离约束的情况下，模拟确实发现了MBP的APO构象。包含tmFRET导出的距离的模拟迅速采用了APO状态。 我们的结论是，tmFRET可以用来驱动蛋白质结构的构象折叠的精度为几埃。 我们继续开发这种方法应用于其他蛋白质。 我们已经探索了包括锌和钴在内的新金属在这些系统中的结合和荧光效应。我们还开发了这些系统的埃级结构的新结构模型，以及如何使用它们来绘制体外或体内其他蛋白质的图谱。这些系统已经在活细胞中进行了测试。因此，tmFRET可以应用于活细胞环境中的蛋白质，以在埃尺度上绘制体内蛋白质的构象动力学。