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。 为了能够通过NMR研究更大的复合物，我们在实验室中开发并采用了新技术。我们采用了新的NMR实验来研究更大的蛋白质以及标记程序（氘代），这是我们小组以前没有的。此外，我们通过仔细的实验和对照表明，除了化学位移之外，还可以使用标量耦合来绘制分子相互作用。这使得人们能够探测变构过程，这在以前很难单独从化学位移信息中区分出来。同时，我们还表明，通过使用顺磁自旋标记，可以探测弱的和瞬态的相互作用。我们举例说明了一种方案，其中可以单独使用顺磁性标记从已知的游离或apo构象确定氨基酸结合蛋白，谷氨酰胺结合蛋白（GlnBP）的结合构象。我们进一步表明，在谷氨酰胺游离形式的GlnBP的情况下，使用广泛的顺磁弛豫增强数据，不采样溶液中的紧密构象。这与麦芽糖结合蛋白形成对比，麦芽糖结合蛋白似乎在溶液中以5%的群体瞬时采样其封闭构象。基于我们在这里所学到的，我们可以扩展这个协议，看看各种弱相互作用的蛋白质参与细胞信号级联。 同时，我们还观察到由于顺磁标记的存在，标量耦合的独特变化。我们认为，一个贡献是由于自旋轨道的极化，由于在核的电子场。此外，也不能排除电子偶极和核偶极之间的干涉通过弛豫机制的贡献。这是目前正在进行的项目，以测试是否可以利用这一结构信息。

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. In order to be able to study larger complexes by NMR we developed and adapted new technology in the laboratory. We adopted new NMR experiments to study larger proteins as well as labeling procedures (deuteration) that was not previously available for our group. In addition we showed by careful experiments and controls that one can use scalar coupling in addition to chemical shift to map out molecular interactions. This enables one to probe allosteric process that was previously difficult to distinguish from chemical shift information alone. At the same time we also showed that thru the use of paramagnetic spin label one can probe weak and transient interaction. We illustrated a protocol where one can determine a bound conformation of an amino acid binding protein, glutamine binding protein (GlnBP), from a known free or apo conformation using paramagnetic label alone. We further showed in the case of glutamine free form of GlnBP, using extensive paramagnetic relaxation enhancement data, does not sample the close conformation in solution. This is in contrast to Maltose binding protein that seems to transiently sample its close conformation, with 5% population, in solution. Based on what we learned here we can extend this protocols to look at various weak interactions in proteins involved in cell signaling cascades. In parallel we also observed a unique change of scalar coupling due to the presence of paramagnetic label. We believe that one contribution is due to the polarization of the spin orbital due to the electron field at the nucleus. In addition a contribution thru relaxation mechanism due to the interference between the electron dipole and nuclear dipole can not be ruled out. This is currently an on going project to test whether one can take advantage of this for structural information.