STRUCTURE DETERMINATION OF A PROKARYOTIC CYCLIC NUCLEOTIDE-MODULATED ION CHANNEL

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。 迄今为止，几乎没有膜蛋白具有可用的高分辨率3D结构。我们已经鉴定并功能性表征了真核环核苷酸门控通道的原核同源物（Nimigean等人，2004，JGP），对于该族没有可用的原子分辨率结构。真核细胞的CNG和HCN通道是信号转导的关键参与者。CNG通道在视觉和嗅觉信号转导中起重要作用，而HCN通道是心脏和大脑中的起搏通道。这种离子通道类型的高分辨率结构将是该领域的重大进展。在过去的一年里，我们一直致力于进一步表征原核通道同源物，除了试图获得天然蛋白质和各种构建体/突变体的衍射质量晶体。我们已经获得了晶体，我们测试了以前在家庭来源和同步加速器光束。衍射质量在家庭来源（7-20 A分辨率）是非常差的，并在同步加速器光束（3.5-8 A）显着改善。不幸的是，到目前为止，数据的质量使我们无法解决原子结构。我们认为我们现在已经通过使用稍微改变的构造优化了这些晶体。

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. To date there are few membrane proteins with available high-resolution 3D structure. We have identified and functionally characterized a prokaryotic homolog of eukaryotic cyclic nucleotide-gated channels (Nimigean et al., 2004, JGP), for which family there is no atomic resolution structure available. The eukaryotic CNG and HCN channels are crucial players in signal transduction. The CNG channels play important roles in visual and olfactory signal transduction while the HCN are the pacemaker channels in the heart and brain. A high-resolution structure of this ion channel type will be a significant advance in the field. We have been working for the past year on further characterizing the prokaryotic channel homolog in addition to trying to obtain diffraction quality crystals of the native protein and various constructs/mutants. We have obtained crystals that we tested previously both at home sources and at synchrotron beams. The diffraction quality was extremely poor at home source (7-20 A resolution) and improved significantly at synchrotron beams (3.5-8 A). Unfortunately, so far the quality of the data precluded us from solving the atomic structure. We think we have now optimized these crystals by using a slightly altered construct.