Dynamin: Ligand Interactions and Conformational Dynamics

发动蛋白（Dynamin）是一种大分子量（~ 98 kDa; 870个氨基酸）的GTP酶，其是一种重要的组分， 对细胞活力至关重要的各种生物过程。在哺乳动物中，发动蛋白 由三个传统的动力蛋白基因（Dyn 1，Dyn 2，Dyn 3）。Dyn 1在神经元中表达，Dyn 2在 Dyn 3在睾丸中表达最高，但在神经元中也可检测到， 肺。纯化的重组发动蛋白可以自组装成环状和螺旋状，这表明所有的 必需的结合相互作用包含在蛋白质序列内。动力素1和2都存在于 涉及单体和多聚体物种的平衡，我们假设，这种低阶 寡聚化可能代表一个新的调节位点。在这个提议中，我们计划使用荧光 偏振和荧光相关光谱（PCS）研究发动蛋白在体外的自缔合 并确定结合的核苷酸和脂质对这种平衡的影响。我们还将扩展这些 通过利用发动蛋白的EGFP构建体研究体内条件。我们将使用PCS来检测 结合的发动蛋白和内亲素在体内，并研究各种突变的影响（在两个发动蛋白 和内啡肽）在这个协会上。早些时候，我们使用了一个巨大的单层囊泡系统（GUV）， 观察荧光标记的发动蛋白与不同脂质系统的相互作用。这些研究 建议的价值GUV系统的动力蛋白与磷脂的相互作用的直接信息。 为了继续这些研究，我们建议使用PCS来研究用小荧光团标记的发动蛋白2， 以及与GUV相关的EGFP构建体。我们进一步假设，GTP释放减慢，当 发动蛋白与支架结合，例如，阴离子脂质体或微管。为了验证这一 我们可能会使用停流动力学方法来测量核苷酸的结合和释放， PIP 2/PC脂质体的存在。 这些研究将提供动力蛋白的强度和意义的定量信息， 以及构象动力学的各个方面。由于发动蛋白是生物多样性和 关键的生物学功能，包括受体介导的内吞作用（参与胆固醇摄取）， 突触囊泡再循环（在神经传导中很重要），这一信息对公众健康很重要。

Dynamin is a large molecular weight (~98kDa; 870 amino acids) GTPase, which is an important component of a wide variety of biological processes which are critical for cell viability. In mammals, dynamin is encoded by three conventional dynamin genes (Dyn1, Dyn2, Dyn3). Dyn1 is expressed in neurons, Dyn2 is ubiquitously expressed, and Dyn3 is most highly expressed in testes but is also detectable in neurons and lung. Purified recombinant dynamin can self-assemble into rings and spirals, which suggests that all the necessary binding interactions are contained within the protein sequence. Both dynamins 1and 2 exist in equilibria involving monomeric and multimeric species and we hypothesis that this lower-order oligomerization may represent a novel site of regulation. In this proposal, we plan to use fluorescence polarization and fluorescence correlation spectroscopy (PCS) to study the self-association of dynamin in vitro and to determine the effects of bound nucleotide and lipid on this equilibrium. We shall also extend these studies to in vivo conditions by utilizing an EGFP construct of dynamin. We shall use PCSto detect association of dynamin and endophilin in vivo and to study the effects of various mutations (in both dynamin and endophilin) on this association. Earlier, we used a giant unilammelar vesicle system (GUV) to directly observe the interaction of fluorescently-labeled dynamin with different lipid systems. These studies suggested the value of the GUV system for direct information on the interaction of dynamin with phospholids. To continue these studies, we proposeto use PCSto to study dynamin 2 labeled with small fluorophores as well as EGFP-constructs, associating with GUVs. We further hypothesize that GTP release is slowed when dynamin is bound to a scaffold, e.g., anionic liposomes or microtubules, prior to polymerization. To test this possibility we will use stopped-flow kinetic methods to measure nucleotide binding and release in the presence of PIP2/PC liposomes. These studies will provide quantitative information on the strengths and significance of dynamin's interactions.and on aspects of its conformaitonal dynamics. Since dynamin is fundamental to diverse and critical biological functions, including receptor mediated endocytosis (involved in cholesterol uptake) and synaptic vesicle recycling (important in nerve conduction), this information is significant to public health.