Imaging the structure and dynamics of membrane proteins

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，我们专门使用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HOLO状态移动到APO状态。在没有tmFRET衍生的距离约束的情况下，模拟没有发现MBP的APO构象。然而，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。 目的2 细胞膜蛋白存在于复杂的分子环境中。例如，许多膜蛋白组装成复合物。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。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的结构，它们的复合物如何组装，以及这些复合物的结构如何在活细胞内调节。 为此，我们一直在观察质膜t-SNARE突触融合蛋白的结构和构象动力学。这种膜蛋白是负责将突触囊泡与质膜融合的蛋白质机制的核心组分。已经提出突触融合蛋白采用封闭的非活性构象和开放的活性构象。为了理解这些转变，我们成像的结构syntaxin 1A在活细胞膜与荧光共振能量转移（FRET）。具体而言，荧光标记的突触融合蛋白1A和膜驻留FRET受体dipicrylamine（DPA）之间的FRET被用来映射突触融合蛋白1A的结构域和质膜的平面之间的相对距离。我们的研究结果映射在这两个国家相对于膜的突触融合蛋白的架构，并打开了大门，以确定在一个复杂的细胞环境与FRET的膜蛋白的结构和结构转换。

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 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 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 specifically 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 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 HOLO state to the APO state. Without tmFRET-derived distance constraints, the simulations did not find the APO conformation of MBP. Simulations that contained the tmFRET-derived distances, however, 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. Aim 2 Membrane proteins in cells exist in a complex molecular environment. For example, many membrane proteins assemble as complexes. Furthermore, dozens of binding partners may transiently interact with membrane proteins to modulate their behavior. 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 structure of these complexes is regulated within living cells. In this aim, we have been observing the structure and conformational dynamics of the plasma membrane t-SNARE syntaxin. This membrane protein is a core component of the protein machinery responsible for fusing synaptic vesicles with the plasma membrane. Syntaxin has been proposed to adopt a closed inactive conformation and an open active conformation. To understand these transitions, we imaged the structure of syntaxin 1A in living cell membranes with fluorescence resonance energy transfer (FRET). Specifically, FRET between fluorescently-tagged syntaxin 1A and the membrane-resident FRET acceptor dipicrylamine (DPA) was used to map the relative distances between domains in syntaxin 1A and the plane of the plasma membrane. Our results map the architecture of syntaxin in both of these states relative to the membrane and have opened the door to determining the structures and structural transitions of membrane proteins in a complex cellular environment with FRET.