Calcium regulatory proteins in plants and yeasts; an NMR approach

钙作为次生信使在真核细胞中起着重要作用。细胞质内Ca2+水平的增加可以通过与钙调蛋白（calmodulin, CaM）等调节性钙结合蛋白结合而起到触发作用。通过结合Ca2+，这些“钙传感器”蛋白可以改变细胞的生理状态。在植物细胞质Ca2+水平增加响应各种非生物和生物胁迫，如寒冷，干旱，盐，伤害或植物病原体的存在。因此，Ca2+是植物胁迫反应的重要介质。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传感器蛋白，如独特的CaM's，许多CaM样蛋白以及各种蛋白激酶和其他具有自身集成的CaM样蛋白结构域的系统。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），这在其他生物中是找不到的。我的研究小组一直对绘制参与植物应激反应的钙传感器蛋白质和蛋白质结构域的分子细节感兴趣。我们在这个研究项目中所依赖的主要实验技术是核磁共振（NMR）光谱，它使我们能够获得这些蛋白质及其复合物在自然溶液状态下的详细结构和动态信息。此外，我们还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。迄今为止，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复合物的性质。在不久的将来，我们希望研究三种直接参与植物胁迫反应的cam样蛋白。此外，我们计划研究钙- cam如何通过结合这些通道的调节细胞质结构域来抑制允许Ca2+流入植物细胞的环核苷酸门控通道。此外，我们将重点关注植物（以及相关的酵母）纤维蛋白，这是一组在植物生长中起重要作用的蛋白质。这些蛋白在进化过程中高度保守，它们由两个钙结合基元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转运蛋白。这种蛋白特异性地将ATP-Mg2+转运到线粒体内部，并通过位于跨膜通道顶部的独特的cam样结构受到Ca2+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.