New Approaches to Protein Structure Determination Using NMR Spectroscopy

自从首次测定蛋白质的结构以来，近50年来，我们对生物大分子在溶液中的三维状态的理解有了很大的提高。现在已经确定，蛋白质在溶液中存在各种不同的状态，其中许多是高度构象异质性的。即使在其天然状态下，蛋白质也会不断经历结构波动，时间尺度从皮秒到秒甚至更长;这些动力学与生物学相关，并影响各种各样的过程，包括酶催化，配体结合和生物分子复合物的形成。人们也越来越认识到，非天然和天然未折叠状态在分子和细胞生物学的许多方面发挥着至关重要的作用。这些状态还包括在蛋白质生物合成和降解过程中出现的那些状态、由固有非结构化肽和蛋白质填充的那些状态、在蛋白质折叠过程中采样的中间体和过渡状态、以及在一系列神经变性和全身性病症（诸如阿尔茨海默病和帕金森病以及II型糖尿病）中涉及的各种致病性错误折叠的多聚体种类。这类状态对结构测定提出了巨大的挑战，因为在许多情况下，它们具有固有的灵活性和构象高度异质性，有几个因素阻碍了对表现出高度结构异质性的蛋白质状态的实验表征。该提案旨在建立一个通用程序，用于确定蛋白质结构的解决方案，利用NMR化学位移和残余偶极耦合（RDC）提供的信息。有几个因素支持这种方法的新奇和可行性。首先，化学位移和RDC代表容易获得的NMR观测值，可以在广泛的条件下测量。此外，RDC是溶液中动力学的高度精确探针。这种方法不仅可以扩展的限制，在系统的大小和准确性的动力学描述，常规就业的NMR在折叠蛋白质的研究，但将提供一个前所未有的工具，用于表征结构异质状态的solution.The方法在这里提出的有可能揭示在很大程度上未开发的蛋白质科学领域，涉及蛋白质在高度异质状态。到目前为止，由于不可能在原子级分辨率下进行系统的实验，传统技术无法表征这种状态。因此，我们希望所提出的方法将提供解决分子和细胞生物学中许多悬而未决的问题的手段。我们设想的一些应用将在蛋白质错误折叠和聚集领域，这是与从神经退行性疾病到系统性淀粉样变性的40多种病理条件相关的过程。因此，这种方法将对制药和生物技术研究以及与蛋白质科学相关的一系列学科（包括化学，物理学和生物学）产生重大影响。

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.