Calcium regulatory proteins in plants and yeasts; an NMR approach

钙在所有真核细胞中作为第二信使起着重要的作用。细胞内钙离子水平的升高可以通过与钙调素(CaM)等调节钙结合蛋白的结合而起到触发作用。总而言之，一旦结合了钙离子，这些“钙感受器”蛋白就可以改变细胞的生理状态。在植物中，细胞内钙离子水平升高是对各种非生物和生物胁迫的响应，如寒冷、干旱、盐、伤害或植物病原体的存在。因此，Ca~(2+)是植物逆境反应的重要中介。最近的基因组测序工作表明，所有植物都含有大量不同的钙敏感蛋白，如不同的CaM，大量的CaM样蛋白以及各种蛋白激酶和其他系统，它们拥有自己的完整的CaM样蛋白结构域。例如，植物(和原生动物)含有一类独特的钙结合蛋白激酶(通常称为CDPK和CCaMK)，这是其他生物体中没有的。我的研究小组一直对绘制参与植物胁迫反应的钙感受器蛋白和蛋白结构域的分子细节感兴趣。我们在这项研究计划中依赖的主要实验技术是核磁共振(核磁共振)波谱，它使我们能够获得这些蛋白质及其天然溶液状态下的络合物的详细结构和动态信息。此外，我们利用各种其他生物物理工具来研究蛋白质-钙和蛋白质-蛋白质相互作用的动力学和能量学。到目前为止，我们已经确定了各种植物蛋白激酶的CaM样结构域的结构，并研究了植物CaM及其与各种酶和跨膜钙泵的络合物的性质。在不久的将来，我们想要研究三个直接参与植物逆境反应的CaM类蛋白。此外，我们计划研究钙-CaM如何通过与这些通道的调节胞质结构域结合来抑制环核苷酸门控通道，从而允许钙离子进入植物细胞。此外，我们将重点关注植物(和相关的酵母)菌毛蛋白，这是一组在植物生长中发挥重要作用的蛋白质。这些蛋白质在进化过程中高度保守，它们由两个钙结合基序和四个肌动蛋白结合的CH结构域组成。最后，我们计划研究一种嵌入线粒体周围膜中的运输蛋白。这种蛋白将ATP-Mg2+特异性地转运到线粒体内部，并通过位于跨膜通道顶部的独特的类CaM结构受到钙离子的调节。通过这些项目获得的知识可能有助于未来提高重要作物的抗旱性或耐盐性，或者可以帮助我们以合理的方式开发新的除草剂。

Calcium plays an important role as a secondary messenger in all eukaryotic cells. An increase in the cytosolic Ca2+ level can act like a trigger by binding to regulatory calcium-binding proteins, such as calmodulin (CaM). Collectively upon binding Ca2+ these 'calcium-sensor' proteins can redirect the physiological status of a cell. In plants the cytosolic Ca2+ level increases in response to various abiotic and biotic stresses such as cold, drought, salt, wounding or the presence of plant pathogens. As such Ca2+ is an important mediator of the plant stress response. Recent genome sequencing efforts have shown that all plants contain a large diversity of calcium-sensor proteins such as distinct CaM's, numerous CaM-like proteins as well as a variety of protein kinases and other systems that possess their own integrated CaM-like protein domains. For example, plants (and protozoa) contain a unique class of calcium binding protein kinases (commonly known as the CDPK's and CCaMK's) that are not found in other organisms. My research group has been interested in mapping out the molecular details of the calcium-sensor proteins and protein domains that participate in the plant stress response. The main experimental technique that we rely upon in this research program is Nuclear Magnetic Resonance (NMR) spectroscopy, which allows us to obtain detailed structural and dynamic information about these proteins and their complexes in their natural solution state. In addition we make use of various other biophysical tools to study the kinetics and energetics of the protein-calcium and protein-protein interactions. To date, we have determined the structures of the CaM-like domains of various plant protein kinases, as well as studied the properties of plant CaMs and their complexes with various enzymes and transmembrane calcium pumps. In the near future we want to study three selected CaM-like proteins that are directly involved in the plant stress response. Moreover, we plan to study how calcium-CaM can inhibit cyclic-nucleotide gated channels, that allow Ca2+ influx in the plant cells, by binding to a regulatory cytoplasmic domain of these channels. In addition, we will focus our attention on plant (and related yeast) fimbrins, a group of proteins that play an important role in plant growth. These proteins have been highly conserved during evolution and they are made up two calcium-binding motifs as well as four actin-binding CH-domains. Finally we plan to study a transport protein that is embedded in the membranes that surround the mitochondria. This protein specifically transports ATP-Mg2+ into the mitochondrial interior and it is regulated by Ca2+ through a unique CaM-like structure, that resides on top of the transmembrane channel. The knowledge gained through these projects may help in the future to improve the drought or salt-resistance of important crops or can help us develop new herbicides in a rational manner.**