Nmr Studies Of The Regulation Of Cell Signaling

GTP结合蛋白、其受体和其在质膜上的效应物之间的相互作用被很好地表征。相比之下，高尔基体膜中类似系统的特定相互作用和功能仍然不清楚。选择G α相互作用蛋白（GAIP）作为研究这种相互作用的模型。GAIP特异性地与Gai 3相互作用，Gai 3已经定位于高尔基体膜。构建了含有GAIP核心结构域（150个残基）的质粒构建体。GAIP的核心结构域包含在G蛋白信号调节剂（RGS蛋白）的新家族中发现的同源结构域。已经使用NMR光谱测定了人GAIP的三维折叠。全球会计和投资方案结构的完善工作正在进行。浓度高于0.1 mM的人GAIP在溶液中以二聚体形式存在。这导致大约34 kD的有效MW。在没有进一步氘化蛋白质的情况下测定初始折叠，这通常是通过溶液NMR进行的这种大小的蛋白质的结构测定。还使用NMR进行了人GAIP的主链动力学研究。这证实了我们的发现，GAIP在溶液中以二聚体形式存在，至少在浓度高于0.1mM时。动态数据还揭示了具有灵活性的区域。GAIP和RGS 4与Gai 1复合的X射线结构的初步比较揭示了与G蛋白结合后的一些构象变化。动态数据表明，可能的灵活性，允许在结构中的构象变化。一个平行的项目，以表达Gal 3亚基已经启动。目的是能够在体外重建人GAIP及其G蛋白补体，并观察其生化特性。我们完成了人GAIP的溶液结构，并与RGS 4与Galpha-i复合物的结构进行了详细的比较。我们的结论是，RGS蛋白的催化活性是通过稳定的复合物结构，而不是通过直接相互作用的RGS的活性位点的Ga。此外，我们已经表明，螺旋V和VI之间的环接触的G α的结构不同，只有N-末端部分。该环的C-末端部分在结合Ga α时不采用不同的构象。我们正在完成该蛋白的动态研究。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。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。我们已经成功地表达了该蛋白，并进行了钙结合以及肽结合的实验。所用肽代表Gai的C-末端螺旋。到目前为止，我们的研究结果表明，蛋白质经历了一定程度的交换两种构象之间的结果在扩大的共振信号。然而，我们已经能够建立钙结合以及肽结合的特异性。我们目前正在鉴定同时存在的不同构象。有趣的是，这种交换过程似乎不影响CALNUC结合钙或其靶肽的能力。我们正在收集NMR数据，以确定在钙存在下人CALNUC的溶液结构。我们已经完成了该蛋白的二级结构测定。我们还完成了测定CALNUC三维结构的数据收集。同时，我们表达15 N和13 C标记的Gai 3，以进行Gai 3在分子的各种功能状态下的结构和动力学研究。到目前为止，我们已经能够表达蛋白质，目前正在研究纯化方案，以提供合适的样品在NMR条件下进行研究。此外，我们已经开始表达AGS 3。它是一种含有几个goLoco结构域的蛋白质。Ags 3似乎具有与RGS结构域相反的功能，即它减缓Gai进入其非活性状态的周转。这种蛋白质的研究将提供一个更广泛的图片的所有可能的结合模式的Ga在其功能，以响应各种细胞信号。

The interaction between the GTP-binding protein, its receptor, and its effector at the plasma membrane is well characterized. In contrast, the specific interaction and function of similar systems in the Golgi membranes is still not clear. A G Alpha Interacting Protein (GAIP) was chosen as a model to study this interaction. GAIP interacts specifically with the Gai3 which has been localized to the Golgi membranes. A plasmid construct containing the core domain (150 residues) of GAIP was constructed. The core domain of GAIP contains a homology domain found in a novel family of regulators of G protein signaling (RGS proteins). The three dimensional fold of human GAIP has been determined using NMR spectroscopy. The refinement of the structure of GAIP is in progress. Human GAIP at a concentration higher than 0.1 mM exists as a dimer in solution. This results in an effective MW of roughly 34 kD. The initial fold was determined without further deuteration of the protein which is typically done for structure determination of proteins of this size by solution NMR. A backbone dynamic study of human GAIP has also been carried out using NMR. This confirms our finding that GAIP exists as a dimer in solution, at least at concentrations higher than 0.1mM. The dynamic data also reveals the regions which have flexibility. Initial comparison of GAIP and the X-ray structure of RGS4 complexed to Gai1 reveals some conformational changes upon binding to the G protein. The dynamic data suggests possible flexibility that allows the conformational change in the structure. A parallel project to express the G ai3 subunit has been initiated. The goal is to be able to reconstruct human GAIP and its G protein complement in vitro and observe the biochemical properties. We have completed the solution structure of human GAIP and carried out detailed comparison to the structure of RGS4 complexed to Galpha-i. We concluded that the activation of catalysis by RGS protein is through stabilization of the complex structure, not by direct interaction of RGS to the active site of Galpha. Furthermore, we have shown that the loop between helix V and VI which contacts the Galpha differs in structure only for the N-terminal portion. The C-terminal portion of this loop does not adopt a different conformation upon binding the Galpha. We are finishing the dynamic study of this protein. We have also initiated a structural study of a calcium binding protein, CALNUC. This protein in the calcium loaded state binds Galpha 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 have succesfully expressed the protein and have carried out experiments on calcium binding as well as peptide binding. The peptide used represents the C-terminal helix of the Gai. Our results so far show that the protein undergoes a certain degree of exchange between two conformations that results in broadening of the resonance signals. However, we have been able to establish the specificity of calcium binding as well as peptide binding. We are currently identifying the different conformations that simultaneously exist. It is interesting that this exchange process does not seem to effect CALNUC's ability to bind calcium or its target peptide. We are collecting NMR data to determine the solution structure of human CALNUC in teh presence of calcium. We have finished the secondary structrue determination of this protein. We also have completed collection of data for the determination of the three dimensional structure of CALNUC. At the same time we are expressing 15N and 13C labeled Gai3 to carry out structural as well as dynamic studies of the Gai3 in the various functional states of the molecule. So far we have been able to express the protein and currently are working on a purification protocol to provide suitable sample for study under NMR condition. In addition we have started the expression of AGS3. It is a protein that contains several goLoco domains. Ags3 seems to have an opposite function than RGS domain, that is it slows the turnover of the Gai into its inactive state. The study of this protein will provide a wider picture of all possible binding modes of the Ga in its function to response to various cell signals.