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首页> 外文学位 >Investigation of the solution and membrane-bound structure of synaptotagmin 1 via electron paramagnetic resonance spectroscopy.
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Investigation of the solution and membrane-bound structure of synaptotagmin 1 via electron paramagnetic resonance spectroscopy.

机译：通过电子顺磁共振波谱研究突触结合蛋白1的溶液和膜结合结构。

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摘要

Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca2+-sensor in neuronal exocytosis. Site-directed spin labeling (SDSL) was used to investigate the conformation and membrane-binding properties of syt1.;First, the Ca2+-dependent membrane interactions of a water soluble fragment of syt1C2AB that contains its two C2 domains (C2A and C2B) were determined using SDSL. Depth parameters were obtained for spin labeled mutants of C2AB when bound to negatively-charged membranes, and distance constraints were used to generate a model for the orientation and position of syt1 on the bilayer interface. Both C2A and C2B penetrate the membrane interface with their first and third Ca2+-binding loops. The two domains are positioned deeper into the bilayer interior when present in the tandem construct. These data indicate that C2A and C2B do not act independently, but influence their mutual membrane penetration.;Second, the Ca2+-independent membrane interactions of syt1C2AB were characterized. It is shown that C2B binds to negatively-charged membranes in a Ca2+- independent manner. SDSL was used to obtain bilayer depth restraints and a simulated annealing routine was used to generate a model for the membrane docking of the syt1C2B(C2A). In this model, the polybasic strand of C2B forms the membrane binding surface for the domain through an electrostatic interaction without penetrating the bilayer. In the presence of Ca2+, the domain rotates from roughly parallel to perpendicular to the bilayer, thus syt1C2AB may act as a switch.;Third, SDSL was used to generate models for the solution and membrane-bound structures of syt1C2AB. In solution, distances between the two C2 domains were measured using double electron-electron resonance (DEER) and were used in a simulated annealing routine. The data indicate that the two C2 domains are flexibly linked, do not interact with each other (with or without Ca 2+) and favor an antiparallel orientation. A similar approach was taken for membrane-associated C2AB, combining both distances and bilayer depth restraints. The restraints only are satisfied when C2A and C2B are antiparallel and docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca2+-dependent manner.
机译：Synaptotagmin 1（syt1）是一种突触小泡膜蛋白，在神经元胞吐作用中充当Ca2 +传感器。使用定点旋转标记（SDSL）来研究syt1的构象和膜结合特性。首先，包含两个C2域（C2A和C2B）的syt1C2AB水溶性片段的Ca2 +依赖性膜相互作用是使用SDSL确定。当绑定到带负电的膜时，获得了自旋标记的C2AB突变体的深度参数，并且使用距离限制条件生成了syt1在双层界面上的取向和位置的模型。 C2A和C2B都通过其第一个和第三个Ca2 +结合环穿透膜界面。当存在于串联结构中时，这两个区域位于双层内部的更深处。这些数据表明，C2A和C2B并不是独立起作用，而是影响它们相互的膜渗透。第二，表征了syt1C2AB的不依赖Ca2 +的膜相互作用。结果表明，C2B以不依赖Ca2 +的方式与带负电荷的膜结合。 SDSL用于获得双层深度约束，模拟退火程序用于生成syt1C2B（C2A）膜对接的模型。在该模型中，C2B的多元链通过静电相互作用形成了结构域的膜结合表面，而没有穿透双层。在存在Ca2 +的情况下，畴从大致平行于垂直于双层的方向旋转，因此syt1C2AB可以充当开关。第三，SDSL用于为syt1C2AB的溶液和膜结合结构生成模型。在溶液中，使用双电子电子共振（DEER）测量了两个C2域之间的距离，并将其用于模拟退火程序中。数据表明，两个C2结构域是柔性连接的，彼此不相互作用（带有或不带有Ca 2+）并且倾向于反平行取向。对于膜相关的C2AB，采用了类似的方法，将距离和双层深度约束结合在一起。仅当C2A和C2B反平行并停靠在相对的双层时，才能满足约束条件。结果表明syt1的功能是以依赖Ca2 +的方式跨过囊泡和质膜表面。

著录项

  • 作者

    Herrick, Dawn Zbell.;

  • 作者单位

    University of Virginia.;

  • 授予单位 University of Virginia.;
  • 学科 Chemistry Biochemistry.;Biophysics General.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 234 p.
  • 总页数 234
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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