The use of paramagnetic tags in structure determination of protein-glycosaminoglycan complexes.

顺磁标签在蛋白质-糖胺聚糖复合物结构测定中的应用。

• 批准号: BB/D020867/1
• 负责人: Dusan Uhrin
• 金额: $ 11.19万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD020867%2F1
• 关键词: use; paramagnetic; tags; structure; determination

Glycosaminoglycans (GAGs) are complex biomolecules that are, in part, built from simple carbohydrates similar to glucose. They form long chains of so-called polysaccharides. GAGs are found on animal (i.e. also human) cell surfaces and extracellular structures and have a wide range of important biological functions. Many of these are realized when a GAG molecule binds to a protein or as we say, forms a GAG-protein complex. Forces that stabilize such complexes are mostly electrostatic, utilizing opposite charges found on GAGs and proteins. As a consequence, when binding happens, oligosaccharides do not insert deep into proteins. Instead, they sit on the protein surface and have only a little contact with the protein they bind. This makes it difficult to determine exactly what such a protein-GAG complex looks like. To make things worse, protein-GAG complexes are often weak and dynamic, with both molecules coming apart frequently. X-ray crystallography and nuclear magnetic resonance spectroscopy (NMR) spectroscopy are the two main experimental techniques that can provide three-dimensional structures of biomolecular complexes. Both methods can offer, in principle, a very detailed picture right down to the level of individual atoms, even for complicated complexes. This is what we want to achieve in the case of protein-GAG complexes. Why do we want to do that? Once we have this information we can investigate the roles of individual atoms in a complex and thus uncover at an atomic level how nature works, or what went wrong when things do not work. We can then pass this information to other researchers who can come up with ideas how to fix or improve things, and design a treatment or a drug. In our research we are proposing to design new NMR spectroscopy techniques so that we can obtain three dimensional structures with atomic resolution also for GAG-protein complexes. This is currently not possible because of the reasons explained above. In order to understand how we want to achieve this, we need to explain briefly how NMR works. In NMR spectroscopy we study the nuclei of atoms. Nuclei behave like small magnets and we know that if there are many magnets close to each other (as there are many nuclei in a protein) they will mutually interact. Without going to any detail, by NMR we can detect if any two magnets, i.e. nuclei, are interacting. If they are, they must be close in space. Therefore once we have established which pairs of nuclei out of thousands present in biomolecular complexes are close to each other, we have in fact determined a three dimensional structure of such complexes. Now we can see why a lack of contact between two interacting molecules and the dynamic nature of such interactions can prevent us from determining structures of protein-GAG complexes: interactions between the magnets from the two molecules are too few and too weak. Fortunately, there is something we can do about it. Unpaired electrons also behave like magnets, but much stronger ones. In fact, approximately 600 times stronger than the strongest magnets of proteins which originate in hydrogen atoms (also called protons). If we can modify GAGs so that they carry an unpaired electron or a stable free radical, as we like to call it, we stand a much better chance of detecting its interactions with protein protons. Therefore, despite the fact that the GAG-protein complexes are loose, weak and dynamic, by studying electron-proton interactions we can determine what their structures look like. In our research we want to develop methods to modify GAGs so that they can carry free radicals, study the binding of such modified molecules to selected, very important GAG-binding proteins and to develop protocols for calculating the structures of these complexes based upon the observation of electron-proton interactions. We believe that our new methods will open new frontiers in the structure determination of protein-GAG complexes in solution.

糖胺聚糖 (GAG) 是复杂的生物分子，部分由类似于葡萄糖的简单碳水化合物构成。它们形成所谓的多糖的长链。 GAG 存在于动物（即人类）细胞表面和细胞外结构中，具有广泛的重要生物学功能。其中许多是在 GAG 分子与蛋白质结合或如我们所说形成 GAG-蛋白质复合物时实现的。稳定此类复合物的力主要是静电力，利用 GAG 和蛋白质上的相反电荷。因此，当发生结合时，寡糖不会深入插入蛋白质中。相反，它们位于蛋白质表面，与它们结合的蛋白质只有很少的接触。这使得很难准确确定这种蛋白质-GAG 复合物是什么样子。更糟糕的是，蛋白质-GAG 复合物通常很弱且动态，两个分子经常分裂。 X射线晶体学和核磁共振波谱（NMR）波谱是可以提供生物分子复合物三维结构的两种主要实验技术。原则上，这两种方法都可以提供非常详细的图像，直至单个原子的水平，甚至对于复杂的复合物也是如此。这就是我们在蛋白质-GAG 复合物中想要实现的目标。我们为什么要这么做？一旦我们掌握了这些信息，我们就可以研究单个原子在复合体中的作用，从而在原子水平上揭示自然是如何运作的，或者当事情不起作用时出了什么问题。然后我们可以将这些信息传递给其他研究人员，他们可以提出如何修复或改进问题的想法，并设计一种治疗方法或药物。在我们的研究中，我们建议设计新的核磁共振波谱技术，以便我们能够获得具有原子分辨率的三维结构，也适用于 GAG-蛋白质复合物。由于上述原因，目前这是不可能的。为了了解我们如何实现这一目标，我们需要简要解释 NMR 的工作原理。在核磁共振波谱中，我们研究原子核。原子核的行为就像小磁铁，我们知道如果有许多磁铁彼此靠近（就像蛋白质中有许多原子核一样），它们就会相互作用。无需了解任何细节，通过核磁共振，我们可以检测任何两个磁铁（即原子核）是否相互作用。如果是的话，它们在太空中一定很接近。因此，一旦我们确定了生物分子复合物中存在的数千个原子核中的哪对彼此接近，我们实际上就确定了此类复合物的三维结构。现在我们可以明白为什么两个相互作用的分子之间缺乏接触以及这种相互作用的动态性质会阻止我们确定蛋白质-GAG 复合物的结构：两个分子的磁体之间的相互作用太少且太弱。幸运的是，我们可以采取一些措施来解决这个问题。不成对的电子也表现得像磁铁，但更强。事实上，它比源自氢原子（也称为质子）的最强蛋白质磁铁强大约 600 倍。如果我们能够修改 GAG，使其携带不成对的电子或稳定的自由基（我们喜欢这样称呼它），我们就有更好的机会检测其与蛋白质质子的相互作用。因此，尽管 GAG-蛋白质复合物是松散的、弱的和动态的，但通过研究电子-质子相互作用，我们可以确定它们的结构。在我们的研究中，我们希望开发修饰 GAG 的方法，使它们能够携带自由基，研究此类修饰分子与选定的、非常重要的 GAG 结合蛋白的结合，并开发基于观察结果计算这些复合物结构的方案电子-质子相互作用。我们相信，我们的新方法将为溶液中蛋白质-GAG 复合物的结构测定开辟新领域。

项目成果

Enabling methodology for the end functionalization of glycosaminoglycan oligosaccharides.

糖胺聚糖寡糖末端功能化的可行方法。

• DOI: http://dx.10.1039/b801666f
• 发表时间: 2008
• 期刊: Molecular bioSystems
• 作者: Gemma E