Imaging the structure and dynamics of membrane proteins

目标 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(双胺)的能量受体，以绘制工程蛋白的构象重排图。这些过渡金属离子FRET(TmFRET)荧光方法与传统FRET方法相比，工作距离更短，使用的染料更小，连接基更短，并且对通常与其他方法相关的取向问题不那么敏感。 在这项工作中，我们使用tmFRET定位了模型蛋白麦芽糖结合蛋白(MBP)在配体结合(Holo)和无配体(APO)状态下的10个独特距离。我们绘制了两种供体染料(一溴双胺和荧光素-5-马来酰亚胺)和两种受体金属(镍和铜)之间的距离。这使得我们总共有40个以MBP为单位的独立距离测量。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射线晶体结构相匹配，精确到几埃。此外，tmFRET能够准确地检测到配体结合过程中蛋白质的结构变化。 有了上述实验数据，我们接下来测试了tmFRET得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，MBP被允许从配体结合状态转移到APO状态。在没有tmFRET衍生距离约束的情况下，模拟确实发现了MBP的APO构象。包含tmFRET导出距离的模拟迅速采用了APO状态。我们得出结论，tmFRET可以用来驱动蛋白质结构的构象折叠，精度达到几埃。 我们一直在继续发展这种方法，就像应用于其他蛋白质一样。我们已经探索了包括锌和钴在内的新金属的结合和荧光效应。此外，我们还开发了这些系统的埃尺度结构的新结构模型，以及它们如何用于在体外或体内映射其他蛋白质。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，在埃尺度上绘制蛋白质在体内的构象动力学图。 随着对蛋白质结构的研究，我们一直在设计和生产一种新型的荧光蛋白。这些蛋白质是基于绿色荧光蛋白(FP)的结构。在这些研究中，我们将过渡金属离子结合位点设计到蛋白质的桶上。这些新的金属结合FP仍然保持其明亮的荧光颜色。然而，在这些新的蛋白质中，有色过渡金属离子(如铜、钴或镍)的结合会导致荧光迅速而强烈的猝灭。我们在体外和培养的哺乳动物细胞中测试了这些新的探针。这些新的探针提供了一种强大的、可调的金属敏感荧光蛋白，可用于过渡金属离子传感应用或用作细胞生物学的多色可调探针。

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. We have additionally 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. Along with these studies of protein structure we have been designing and producing a new type of fluorescence protein. These proteins are based on the structure of the green fluorescent proteins (FPs). In these studies we engineered transition metal ion binding sites onto the barrel of the protein. These new metal-binding FPs still retains their bright fluorescent colors. In these new proteins, however, the binding of colored transition metal ions such as copper, cobalt, or nickel causes rapid and robust quenching of fluorescence. We tested these new probes in vitro and in cultured mammalian cells. These new probes provide a robust and tunable metal sensitive fluorescent protein that can be used for transition metal ion sensing applications or as multi-color tunable probes for cell biology.