New Approaches to Protein Structure Determination Using NMR Spectroscopy

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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.