New Approaches to Protein Structure Determination Using NMR Spectroscopy

使用核磁共振波谱测定蛋白质结构的新方法

• 批准号: EP/G049998/1
• 负责人: Alfonso De Simone
• 金额: $ 28.51万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FG049998%2F1
• 关键词: New; Approaches; Protein; Structure; Determination

In the nearly 50 years since the first determinations of the structures of proteins our understanding of three-dimensional states that biological macromolecules adopt in solution has enormously improved. It is now well established that proteins populate a wide variety of different states in solution, many of which are highly conformationally heterogeneous. Even in their native states proteins constantly undergo structural fluctuations with timescales ranging from picoseconds to seconds and beyond; these dynamics are biologically relevant and influence a wide variety of processes including enzymatic catalysis, ligand binding and the formation of biomolecular complexes. It is also being increasingly recognised that non-native and natively unfolded states play crucial roles in many aspects of molecular and cell biology. These states include also those that appear during protein biosynthesis and degradation, those populated by intrinsically unstructured peptides and proteins, the intermediates and transition states sampled during the protein folding, and the variety of pathogenic misfolded multimeric species implicated in a range of neurodegenerative and systemic disorders, such as Alzheimer's and Parkinson's diseases, and type II diabetes. States of this type pose a formidable challenge for structure determination, because, in many cases, they are inherently flexible and conformationally highly heterogeneous.Several factors hamper the experimental characterization of the states of proteins that exhibit high levels of structural heterogeneity. This proposal aims to establish a general procedure for the determination of protein structures in solution that exploits the information provided by NMR chemical shifts and residual dipolar couplings (RDCs). There are several factors supporting the novelty and feasibility of this approach. First, chemical shifts and RDCs represent readily accessible NMR observables, which can be measured in a wide range of conditions. In addition RDCs are highly accurate probes of the dynamics in solution. Not only this approach can extend the limits, in terms of size of the systems and accuracy of the dynamics description, of the conventional employment of NMR in the study of folded proteins but will provide an unprecedented tool for characterizing structurally heterogeneous states in solution.The method proposed here has the potential to shed light on largely unexplored areas of protein science that involve proteins in highly heterogeneous states. So far, conventional techniques have failed in characterizing such states owing to the impossibility of carrying out systematic experiments at atomistic resolution. We do therefore expect that the proposed method will provide the means for addressing many open questions in molecular and cell biology. Some of the applications that we envisage will be in the field of protein misfolding and aggregation, which are processes that have been associated to over 40 pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. Therefore this approach will have a substantial impact in pharmaceutical and biotechnological research, as well in a range of disciplines related to protein science including chemistry, physicis and biology.

自蛋白质结构的首次确定以来，我们对溶液中生物大分子采用的三维状态的理解已得到了极大的改善。现在已经确定的是，蛋白质在溶液中占多种不同状态，其中许多蛋白质在高度构象上是异质的。即使在其本地状态，蛋白质也不断地经历结构波动，时间尺度从picseconds到秒及以后；这些动力学在生物学上相关，并影响各种过程，包括酶促催化，配体结合和生物分子复合物的形成。越来越多地认识到，非本地和本地展开的状态在分子和细胞生物学的许多方面都起着至关重要的作用。 These states include also those that appear during protein biosynthesis and degradation, those populated by intrinsically unstructured peptides and proteins, the intermediates and transition states sampled during the protein folding, and the variety of pathogenic misfolded multimeric species implicated in a range of neurodegenerative and systemic disorders, such as Alzheimer's and Parkinson's diseases, and type II 糖尿病。这种类型的状态在结构确定方面构成了巨大的挑战，因为在许多情况下，它们本质上是柔性和构象的高度异质性的。几个世纪因素阻碍了表现出较高结构性异质性的蛋白质状态的实验表征。该提案旨在建立一种确定溶液中蛋白质结构的一般程序，以利用NMR化学移位和残留偶极耦合（RDC）提供的信息。有几个因素支持这种方法的新颖性和可行性。首先，化学位移和RDC代表易于访问的NMR可观察物，可以在各种条件下进行测量。另外，RDC是溶液中动力学的高度准确探针。在折叠蛋白的研究中，这种方法不仅可以从系统的大小和动力学描述的准确性方面扩展限制，但将提供一种前所未有的工具，用于在溶液中表征结构上异质的状态。此处提出的方法可以使蛋白质科学的蛋白质科学概述较大的蛋白质范围，从而使蛋白质的蛋白质具有极大的影响。到目前为止，由于无法在原子分辨率上进行系统的实验，传统技术未能表征此类状态。因此，我们确实期望提出的方法将提供解决分子和细胞生物学许多开放问题的手段。我们设想的某些应用将在蛋白质错误折叠和聚集领域，这些过程与从神经退行性疾病到系统性淀粉样蛋白的40多种病理状况相关的过程。因此，这种方法将对药物和生物技术研究产生重大影响，以及与蛋白质科学有关的一系列学科，包括化学，物理学和生物学。

项目成果

Role of hydration in collagen recognition by bacterial adhesins.

水合在细菌粘附素识别胶原蛋白中的作用。

• DOI: 10.1016/j.bpj.2011.03.033
• 发表时间: 2011
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Vitagliano L