Dynamin: Ligand Interactions and Conformational Dynamics

Dynamin是一种大分子量(~98 kDa；870个氨基酸)的GTP酶，是其重要组成部分 对细胞存活至关重要的各种生物过程。在哺乳动物中，动力素是编码的 由3个传统的动力蛋白基因(dyn1、dy2、dy3)决定。动态1在神经元中表达，动态2在神经元中表达 在睾丸中表达最高，但也可在神经元和 阿龙。纯化的重组动力素可以自组装成环和螺旋，这表明所有的 必需的结合作用包含在蛋白质序列中。动力素1和2都存在于 涉及单体和多聚体物种的平衡，我们假设这个低阶 寡聚化可能代表了一个新的调控部位。在这项提案中，我们计划使用荧光 偏振相关光谱和荧光相关光谱研究动力素的体外自结合 并确定结合核苷酸和脂类对这种平衡的影响。我们还将延长这些 通过利用动力素的绿色荧光蛋白构建来研究体内条件。我们将使用PCS来检测 体内动力蛋白和内亲和素的关系以及研究各种突变(在两者中)的影响 和亲内素)。早些时候，我们使用了一个巨大的单囊泡系统(Guv)来直接 观察荧光标记的动力素与不同脂质系统的相互作用。这些研究 提示了GUV系统对动力蛋白与磷脂相互作用的直接信息的价值。 为了继续这些研究，我们建议使用PCSto来研究用小荧光标记的Dynamin 2作为 以及与GUV相关联的EGFP构造。我们进一步假设GTP的释放在以下情况下会变慢 动力蛋白在聚合前结合到支架上，例如阴离子脂质体或微管。为了测试这一点 我们可能会使用停流动力学方法来测量核苷酸结合和释放。 PIP2/PC脂质体的存在。 这些研究将提供有关动力素的优势和意义的定量信息 相互作用。以及它的构象动力学的各个方面。因为动力素是多样化和 重要的生物学功能，包括受体介导的内吞作用(参与胆固醇摄取)和 突触小泡循环(在神经传导中很重要)，这一信息对公共健康具有重要意义。

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.