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。我们试图表达和纯化Gai，以研究其与CALNUC的特异性相互作用。 我们成功地解决了CALNUC的结构。我们发现蛋白质确实结合了2个钙离子。我们还确定了两个结合位点具有相似的结合亲和力。当钙离子被去除时，蛋白质经历解折叠事件。这是钙结合蛋白家族所独有的，我们推测这与蛋白质作为钙信号传导以及缓冲蛋白的功能有关。我们最近确定了CALNUC对Gai 3的C-末端螺旋的亲和力。我们采用偏振各向异性。解离常数相当弱，这与在细胞竞争测定中观察到的一致。我们现在正试图确定对全长Gai 3的亲和力，目的是研究这两种蛋白质复合物中的结构决定因素，这些决定因素定义了它们在信号调节中的作用。 到目前为止，我们已经在体外证明了CALNUC与Gai 3的结合，如果是真的，那一定是非常弱的。我们目前正在努力确定可能调节这种相互作用的可能伙伴的特征。 我们开发的方法，以提高我们的能力，通过NMR表征这种类型的研究。我们研究了J耦合测量以及化学位移中可能的偏差。通常，人们只能测量化学位移张量的平均值，而在溶液中不可能测量全部张量。我们最近开发了一种方法来测量溶液中的化学位移张量。除了我们从NMR测量中获得的原子位置之外，全化学位移张量还为我们提供了对结构环境非常敏感的原子密度分布的变化。我们发现，在羰基化学位移张量的情况下，氢键强度占主导地位的三个张量分量中的两个。而对于酰胺氮，有更多的因素，有助于观察到的张量。我们将扩展这项新技术，以比以前更详细的水平进一步研究蛋白质结构。 去年，我们发明了一种在磁场中排列蛋白质的新方法。我们使用了我们在现场培养的Colagen I型来创建一个有序的基质，我们可以将我们想要研究的蛋白质扩散到其中。这种凝胶材料在很宽的温度和pH值范围内都是稳定的。更重要的是，它在洗涤剂环境下是稳定的，使我们能够研究膜蛋白。此外，我们还进一步研究了化学位移张量。我们决定把注意力集中在张量的变化上，而不是绝对值。这样我们就可以分离出对化学位移张量变化的单一结构贡献。我们使用谷氨酰胺结合蛋白作为模型系统。我们使用谷氨酰胺结合形式的蛋白质和游离形式之间的变化。这使我们能够研究氢键、二面角和堆积对化学位移张量的影响。

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 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 express and purify Gai to study its specific interaction with CALNUC. We succesfully solved the structure of CALNUC. We showed that the protein does bind 2 calciums. We also determined that both bonding sites have similar binding affinity. The protein undergoes an unfolding event when the calciums are removed. This is unique for calcium binding protein family and we hypothesize that this is correlated to the function of the protein as calcium signaling as well as buffering protein. We recently determined the affinity of CALNUC towards the C-terminal helix of Gai3. We employed polarization anisotropy. The dissociation constant is quite weak which is in agreement with what has been observed in cell competition assays. We are now trying to determine the affinity towards the full length Gai3, with the goal of studying structural determinants in the complex of these two proteins that define their role in signal regulation. So far we have shown in vitro that the binding of CALNUC to Gai3 if it is true must be very weak. We are currently trying to characterize possible partners that might regulate this interaction. We developed methods to improve our ability to characterize this type study by NMR. We looked into possible deviations in J coupling measurements as well as chemical shifts. Typically one only measures the average of the chemical shift tensor and the full tensor was not possible to be measured in solution. We recently developed a way to measure chemical shift tensors in solution. In addition to atomic position that we obtained from NMR measurements the full chemical shift tensor provides us with variation in the atomic density distribution that is very sensitive to structural environment. We showed that in the case of carbonyl chemical shift tensor, hydrogen bond strength dominates two of the three tensor components. While for amide nitrogen, there are a lot more factor that contribute to the observed tensor. We will expand on this new technology to further study protein structure at a much more detailed level than previously possible. This previous year we invented a new method to align protein in a magnetic field. We used Colagen type I that we polymerize in the field to create a well ordered matrix into which we can diffuse protein that we would like to study. This gel material is table at very wide range of temperature and pH. More importantly it is stable under detergent environment allowing us to study membrane proteins. In addition we went further in studying chemical shift tensor. We decided to concentrate on changes of the tensor rather than the absolute values. This way we can isolate a single structural contribution to the chemical shift tensor changes. We used Glutamine Binding protein as a model system. We used the changes between the glutamine bound form of the protein and the free form. This has allowed us to look at hydrogen bond, dihedral angle, and packing influences on chemical shift tensor.