SYNAPTOTAGMIN 1

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Synaptotagmin 1是一种突触囊泡蛋白，已被证明可作为触发快速神经递质释放的Ca 2+传感器。突触结合蛋白1的大部分胞质区域由两个C2结构域形成，称为C2 A结构域和C2B结构域。我们以前已经解决了两个synaptotagmin 1 C2-域的三维结构，并表明它们通过β-三明治尖端的环结合多个Ca 2+离子。这些环也介导钙离子依赖性结合带负电荷的磷脂，最有可能的活动，触发释放的突触结合蛋白1的功能的基础。这些结果导致了C2 A结构域和C2B结构域的顶部环与膜接触并在磷脂结合中合作的模型。 一个片段含有两个突触结合蛋白1 C2-域（C2 AB片段）是单体在溶液中的存在或不存在的Ca 2+在蛋白质浓度接近1 mM。在这些研究的过程中，动态光散射实验表明，C2 AB片段诱导囊泡聚集后Ca 2+依赖性结合，这种集群可以通过添加EGTA逆转。我们的第一个目标是可视化使用cryo-EM突触结合蛋白的囊泡聚集活性。 最近报道，C2 AB片段形成七聚体寡聚体后，钙依赖性磷脂结合。这些低聚物通过电子显微镜（EM）观察，对沉积在EM网格上的脂质单层结合的C2 AB片段样品进行负染色。然而，我们还没有能够观察到这样的低聚物的化学交联或荧光共振能量转移实验。该项目的第二个目的是，看看是否synaptotagmin是单体的磷脂膜上，或者如果它形成七聚体的环状寡聚体在电子显微镜观察单层。我们还想测量两个聚集的囊泡之间的距离，以计算囊泡之间的空间是否足够大，以容纳直径为11 nm的突触结合蛋白寡聚体。 该系统提供了研究神经功能，神经递质释放和膜融合方面的机会。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Synaptotagmin 1 is a synaptic vesicle protein that has been shown to act as the Ca2+ sensor that triggers fast neurotransmitter release. Most of the cytoplasmic region of synaptotagmin 1 is formed by two C2-domains that are referred to as C2A-domain and C2B-domain. We have previously solved the three-dimensional structures of both synaptotagmin 1 C2-domains and shown that they bind multiple Ca2+ ions through loops at the tip of a beta-sandwich. These loops also mediate Ca2+-dependent binding to negatively charged phospholipids, the activity that most likely underlies the function of synaptotagmin 1 in triggering release. These results have led to a model whereby the top loops of the C2A-domain and C2B-domain are in contact with the membrane and cooperate in phospholipid binding. A fragment containing both synaptotagmin 1 C2-domains (C2AB-fragment) is monomeric in solution in the absence or presence of Ca2+ at protein concentrations close to 1 mM. In the course of these studies, dynamic light scattering experiments suggested that the C2AB-fragment induces vesicle clustering upon Ca2+-dependent binding, and that such clustering can be reversed by addition of EGTA. Our first aim is to visualize this vesicle clustering activity of synaptotagmin using cryo-EM. It was recently reported that the C2AB-fragment forms heptameric oligomers upon Ca2+-dependent phospholipid binding. These oligomers were observed by electron microscopy (EM) performed with negative staining on samples of C2AB-fragment bound to lipid monolayers deposited on EM grids. However, we have not been able to observe such oligomers by chemical cross-linking or fluorescence resonance energy transfer experiments. The second aim of this project is to see whether synaptotagmin is monomeric on the phospholipid membranes or if it forms heptameric ring-like oligomers as observed in electron microscopy of monolayers. We would also like to measure the distance between two clustered vesicles to calculate whether the space between the vesicles is big enough to accommodate synaptotagmin oligomers of 11 nm diameter. This system afford the opportunity to study aspects of neural functioning, neurotransmitter release and membrane fusion in general.