Molecular reaction mechanisms of heterotrimeric G-Proteins

异三聚体G蛋白是细胞内信号通路的中心开关。它们通常通过G蛋白偶联受体(GPCR)被激活，后者催化GDP到GTP的交换。这会导致异三聚体复合体分解成其亚单位，并激活重要的信号通路。它们被Galpha亚基内的GTP水解物再次关闭。GPCRs目前正由世界各地的许多研究小组进行研究，特别是由于它们在药理学中的重要性。相反，我们将使用时间分辨FTIR差异光谱和生物分子模拟，通过广泛的、综合的方法来研究Galpha蛋白的较不详细的研究。Galpha亚基由G结构域和一个附加的全α结构域组成，G结构域在小的GTP酶中也是保守的。从X射线结晶学中获得的Galpha的几种蛋白质结构已经存在。然而，使用的是非水解性GTP类似物，这严重干扰了催化中心。互补的、时间分辨的FTIR光谱可以利用天然核苷酸来解析生理条件下蛋白质的动态以及它与原子细节的相互作用。我们已经成功地将这种方法应用于许多小的GTP酶。在我们的前期工作中，我们能够将这种方法转移到Galpha蛋白上。为了从红外光谱中解码详细的分子信息，还采用了QM/MM计算。在这里，我们想要阐明重要的催化氨基酸的作用。除了谷氨酰胺，稳定亲核水，特别是催化精氨酸的作用将被确定。与小的GTP酶不同，后者是催化中心内的固有残基。此外，我们想要了解RGS-蛋白是如何与小的GTP酶的缺口不同的，能够在不与核苷酸直接相互作用的情况下进一步加速水解的。这调节了信号通路的中断。除了野生型蛋白，我们还想确定精氨酸和谷氨酰胺突变引起的蛋白功能障碍。这些突变在麦克库恩-奥尔布赖特综合征和癌症等严重疾病中发挥着重要作用。我们将从抑制的GI和激活的Gs开始我们的研究。稍后，计划研究GQ和GT，以阐明机制的改变和相似之处。进一步的疾病，如霍乱和百日咳，是由毒素介导的，通过ADP-核糖化修饰Galpha蛋白。这一修饰的机理将被详细研究。

Heterotrimeric G-proteins are central switches within signalling pathways in cells. These are usually switched on via G-protein coupled receptors (GPCR), which catalyse the exchange from GDP to GTP. This causes the breakup of the heterotrimeric complex into its subunits and activates important signalling pathways. They are switched off again by GTP hydrolysis within the Galpha-subunit. GPCRs are currently investigated by numerous research groups worldwide, especially due to their importance in pharmacology. In contrast, we will investigate the less detailed studied hydrolysis of Galpha-proteins by a broad, integrative approach using time-resolved FTIR difference-spectroscopy and biomolecular simulations. The Galpha-subunits consist of the G-domain, conserved also in small GTPases, and an additional all-alpha domain. Several protein structures of Galpha obtained from X-ray crystallography are already available. However, non-hydrolysable GTP analogues were used, which interfere seriously with the catalytic centre. Complementary, time-resolved FTIR-spectroscopy can resolve the dynamics of the protein under physiological conditions and its interaction with atomic detail using the natural nucleotide. We have applied this approach already successfully for many small GTPases. In our preliminary work, we were able to transfer this approach also to Galpha-proteins. In order to decode detailed molecular information from the IR spectra, QM/MM calculations are applied in addition. Here we want to elucidate the role of the catalytically important amino acids. Besides a glutamine, stabilizing the nucleophilic water, in particular the role of the catalytic arginine will be determined. The latter is, in contrast to small GTPases, an intrinsic residue within the catalytic centre. Further, we want to understand how RGS-proteins, unlike GAPs of small GTPases, are able to further accelerate hydrolysis, without direct interaction with the nucleotide. This regulates the interruption of signalling pathways. Besides wildtype protein, we want to determine especially dysfunctions of the protein induced by mutations of the arginine and the glutamine. These mutations play an important role in severe diseases as the McCune-Albright syndrome and cancer. We will start our investigations with the inhibitory Gi and the activating Gs. Later it is planned to investigate Gq and Gt, to elucidate mechanistic alterations and similarities. Further diseases like cholera and pertussis are mediated by toxins, modifying Galpha-proteins by ADP-ribosylation. The mechanism of this modification will be investigated in detail.