Imaging the structure and dynamics of membrane proteins

膜蛋白的结构和动力学成像

• 批准号: 8558038
• 负责人: Justin Taraska
• 金额: $ 56.06万
• 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/8558038
• 关键词: Adopted; Architecture; Behavior; Binding; Biological; Cell membrane; Cells; Complex; Copper; DNA Sequence Rearrangement; Data; Dyes; Environment; Fluorescein; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Image; Ions; Life; Ligand Binding; Ligands; Light; Maleimides; Maps; Measurement; Measures; Membrane; Membrane Proteins; Metals; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Nickel; Post-Translational Protein Processing; Protein Engineering; Proteins; Regulation; Relative (related person); Signal Transduction; Structure; Synapses; Synaptic Vesicles; Testing; Transition Elements; Vesicle; Work; bimanes; dipicrylamine; extracellular; falls; in vivo; maltose-binding protein; molecular dynamics; protein complex; protein structure; receptor; simulation; syntaxin; syntaxin 1A; target SNARE proteins; tool

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.

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，我们专门使用tmFRET来定位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。在这些受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导出距离的模拟很快采用了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。 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。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，以及这些复合体的结构在活细胞内是如何调节的。 为此，我们一直在观察质膜T-SNARE合成素的结构和构象动力学。这种膜蛋白是负责将突触小泡与质膜融合的蛋白质机制的核心组成部分。Synaxin已被提出采用封闭的非活性构象和开放的活性构象。为了了解这些转变，我们用荧光共振能量转移(FRET)成像了活细胞膜中合成素1A的结构。具体地说，利用荧光标记的Synaxin 1A与膜上驻留的FRET受体二匹胺(DPA)之间的FRET来定位Synaxin 1A中的结构域与质膜平面之间的相对距离。我们的结果绘制了Synaxin在这两种状态下相对于膜的结构图，并为用FRET在复杂的细胞环境中确定膜蛋白的结构和结构转变打开了大门。