Nmr Studies Of The Regulation Of Cell Signaling

细胞信号传导调节的核磁共振研究

• 批准号: 6541681
• 负责人: NICO TJANDRA
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6541681
• 关键词: G protein; Golgi apparatus; biological signal transduction; calcium binding protein; conformation; guanine nucleotide binding protein; guanosine triphosphate; guanosinetriphosphatase activating protein; nuclear magnetic resonance spectroscopy; protein folding; protein protein interaction; protein structure function; structural biology

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 structure determination of this protein. 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.

gtp结合蛋白、受体及其在质膜上的效应物之间的相互作用已被很好地表征。相比之下，高尔基膜中类似系统的具体相互作用和功能尚不清楚。选择G α相互作用蛋白（GAIP）作为模型来研究这种相互作用。GAIP与定位于高尔基膜的Gai3特异性相互作用。构建了包含GAIP核心结构域（150个残基）的质粒结构体。GAIP的核心结构域包含一个在G蛋白信号（RGS蛋白）调节因子家族中发现的同源结构域。利用核磁共振光谱测定了人体GAIP的三维折叠。GAIP结构的细化正在进行中。人体GAIP浓度大于0.1 mM时以二聚体形式存在于溶液中。这导致有效MW约为34 kD。最初的折叠是在没有进一步氘化蛋白质的情况下确定的，这通常是通过溶液核磁共振来确定这种大小的蛋白质的结构。利用核磁共振技术对人体GAIP进行了主干动力学研究。这证实了我们的发现，至少在浓度高于0.1mM时，GAIP以二聚体的形式存在于溶液中。动态数据还揭示了具有灵活性的区域。初步比较GAIP和与Gai1络合的RGS4的x射线结构，发现与G蛋白结合后构象发生了一些变化。动态数据表明，可能的灵活性，允许构象变化的结构。一个表示gai3亚基的并行项目已经启动。目的是在体外重建人gaia及其G蛋白补体，并观察其生化特性。我们完成了人类GAIP的解结构，并与RGS4复配galpha - 1的结构进行了详细的比较。我们得出结论，RGS蛋白的催化激活是通过稳定复合物结构，而不是通过RGS与Galpha活性位点的直接相互作用。此外，我们已经证明，只有在n端部分，与α α体接触的螺旋V和螺旋VI之间的环结构不同。该环的c端部分在与Galpha结合时不采用不同的构象。我们正在完成对这种蛋白质的动态研究。我们还启动了钙结合蛋白CALNUC的结构研究。这种蛋白质在钙负载状态下与高尔基体中的Galpha结合。据信，CALNUC通过与Galpha的相互作用来调节高尔基体中的钙浓度。CALNUC似乎不影响Galpha中GTP的水解。因此，我们假设有几种不同的模式结合到Galpha。这些不同的模式控制着脑胼胝体对某种刺激作出反应的不同功能子集。我们构建了包含两个EF手的CALNUC质粒。我们成功表达了该蛋白，并进行了钙结合实验和肽结合实验。所使用的肽代表Gai的c端螺旋。到目前为止，我们的研究结果表明，蛋白质在两种构象之间进行了一定程度的交换，从而导致共振信号的拓宽。然而，我们已经能够建立特异性钙结合以及肽结合。我们目前正在识别同时存在的不同构象。有趣的是，这种交换过程似乎并不影响CALNUC结合钙或其靶肽的能力。我们正在收集核磁共振数据，以确定人类CALNUC在钙存在下的溶液结构。我们已经完成了该蛋白的二级结构测定。同时，我们表达了15N和13C标记的Gai3，以在分子的各种功能状态下对Gai3进行结构和动力学研究。