Nmr Studies Of The Regulation Of Cell Signaling

我们还开始了钙结合蛋白CALNUC的结构研究。该蛋白质在钙负载状态下结合高尔基体中的Ga（Ga）。据信CALNUC通过其与Ga相互作用来调节高尔基体中的钙浓度。CALNUC似乎不影响Galalpha中的GTP水解。因此，我们假设有几种不同的模式结合到α。这些不同的模式支配着阿尔法对某种刺激做出反应的不同功能的子集。我们构建了包含两个EF手的CALNUC质粒。我们现在有了CALNUC的钙结合结构域的结构。它具有典型的钙结合环。我们正在表征其钙结合，并试图将结合亲和力与其结合环结构相关联。这种蛋白质的骨架动力学已经被测量过了，我们正在将其与这种蛋白质的功能联系起来，特别是它的Ga相互作用。我们希望能够从CALNUC的结构中推断出它的具体功能。到目前为止，从我们的钙结合实验中，我们认为它的功能是缓冲钙，因为相对于与信号传导相关的其他钙结合蛋白，它的钙亲和力较低。有趣的是，CALNUC确实与Ga相互作用。我们尝试表达和纯化Gai，以研究其与CALNUC的特异性相互作用。 另一种我们感兴趣的蛋白质是AGS3。这似乎下调Ga对细胞信号传导的响应。到目前为止，我们已经能够表达蛋白质到可接受的水平，然而，我们还没有能够纯化这种蛋白质到可接受的水平内的NMR研究。

We have also initiated a structural study of a calcium binding protein, CALNUC. This protein in the calcium loaded state binds Galpha (Ga) in the Golgi. It is believed that CALNUC is regulated through its interaction with Galpha to modulate calcium concentration in the Golgi apparatus. CALNUC does not seem to effect the GTP hydrolysis in Galpha. Therefore we hypothesize that there are several different modes of binding to the Galpha. These different modes govern a subset of different functions that the Galpha would undertake to respond to a certain stimulus. We have constructed the CALNUC plasmid which encompasses the two EF hands. We now have the structure of the calcium binding domain of CALNUC. it posseses an a typical calcium binding loop. We are characterizing its calcium binding and try to correlate binding affinity to its binding loop structure. The backbone dynamics of this protein has been measured and we're in the process of correlating that to function of this protein, specifically its Ga interaction. We hope to be able to deduce from the structure of CALNUC its specific function. So far from our calcium binding experiments we believe that its function is to buffer calcium, due to the lower calcium afinity relative to other calcium binding proteins that are associated with signaling. Interestingly CALNUC does interact with Ga. We are trying to expressed and purify Gai to study its specific interaction with CALNUC. Another protein that we're insterested in is AGS3. This seems to down regulate Ga response to cell signaling. So far we have been able to express the protein to acceptable level, however we have not been able to purify this protein to within acceptable level for NMR study.