CHROMAGRANIN A COILED-COIL STRUCTURE

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。 嗜铬粒蛋白A（Chromogranin A，CHGA）是神经内分泌颗粒核心的主要结构蛋白。我们最近进行的一些物理和光学实验表明，蛋白质的扩展构象。一个同源性模型现在表明，CHGA可能采取卷曲螺旋结构，很像原肌球蛋白（PDB TPM 1，1c 1g A链）。这种构象可以解释CHGA的许多不寻常的物理性质，并提供了一种神经内分泌颗粒核心内密集蛋白质包装的机制。我们想通过实验测试CHGA是否作为螺旋线圈存在，使用低角度X射线散射。我们手头有纯化的重组的E.大肠杆菌表达的嗜铬粒蛋白A（CHGA）用于这些实验。通过6-His-亲和层析纯化CHGA，并通过SDS-PAGE验证。我们已经将这种材料用于几个物理和生物化学实验，包括圆二色谱，凝胶过滤来分配分子量和斯托克斯半径，以及培养中内皮细胞的内分泌（内皮素释放）活性。在这里，我们只想做低角X射线散射，以确认是否存在螺旋土结构。

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. Chromogranin A (CHGA) is the major structural protein in the core of neuroendocrine secretory granules. Several physical and optical experiments performed recently by us suggest an extended conformation for the protein. A homology model now suggests that CHGA may assume a coiled-coil structure, much like that of tropomyosin (PDB TPM1, 1c1g A chain). Such a conformation would explain manyof the unusual physical properties of CHGA, and provide a mechanism for dense protein packing within neuroendocrine secretory granule cores. We would like to test experimentally whther CHGA does exist as a coiled-coil, using low angle x-ray scattering. We have in hand purified, recombinant, E. coli-expressed chromogranin A (CHGA) for these experiments. CHGA was purified by 6-His-affinity chromatography, and verified by SDS-PAGE. We have already used this materialfor several physical and biochemical experiments, including circular dichroism spectroscopy, gel filtration to assign molecular weight and Stokes radius, and endocrine (endothelin-releasing) activity on endothelial cells in culture. Here we wish only to do low angle x-ray scattering, to confirm whether a coiled-soil structure exists.