New Approaches to Protein Structure Determination Using NMR Spectroscopy

Lead Research Organisation: University of Cambridge
Department Name: Chemistry

Abstract

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.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
EP/G049998/1 01/03/2010 01/07/2011 £223,737
EP/G049998/2 Transfer EP/G049998/1 02/07/2011 01/03/2013 £128,374
 
Description We have developed a new method to use bimolecular NMR to resolve protein structure and dynamics using advanced combinations of experiments and molecular simulations.

The research has not only provided a step forward in the interdisciplinary combination of experiments and theory, but has shown that it is possible to describe accurately the structure and dynamics of proteins, including those states that are intrinsically disordered and exert their function through ensembles of conformations with similar probability of existence.

The research over the three years of the fellowship (which includes EP/G049998/1 and EP/G049998/2) has been so far summarised in 19 articles.
Exploitation Route The research has produced protocols and software that are described in a number of research articles. The software is freely distributed upon request.

Future developments include the generation of a website where the software can be downloaded, including a detailed documentation and examples.
Sectors Healthcare,Manufacturing, including Industrial Biotechology

 
Description The main outcome from EP/G049998/1 and EP/G049998/2 is to provide new tools to resolve structures and dynamics of proteins in an unprecedentedly accurate manner. The impact will therefore arise from the ability to study proteins that cannot be currently characterised using standard techniques of structural biology. In the decades following the pioneering structures of proteins, the information obtained by techniques such as X-ray crystallography or bimolecular NMR has been translated into applications ranging from drug design to biotechnologies. Our new methods are tailored to reach a similar level of exploitation. One of the possible fields, which is of key relevance for the society, is the use of these methods to tackle proteins such as Abeta (currently under investigation in my lab) and alpha synuclein (for which we have recently provided a major contribution based on our methods - Nature Communications, 2014, 5:3827) that are respectively connected to Alzheimer's and Parkinson's diseases. As these biomolecules have huge medical relevance, it is anticipated that the ability to characterise their structural ensembles, i.e. arising from the methods developed in EP/G049998/1 and EP/G049998/2, will provide a key impact on the society by helping to face top medical challenges of our times.
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description University of Cambridge 
Organisation University of Cambridge
Department Department of Applied Mathematics and Theoretical Physics (DAMTP)
Country United Kingdom 
Sector Academic/University 
PI Contribution Our contribution to the partnership is to provide NMR support for structural investigations in biomolecular processes linked to neurodegenerative diseases
Collaborator Contribution The partner's contribution to the collaborative projects is to provide tools to assess the toxicity in vivo of protein aggregates linked to neurodegenerative diseases, which also represents a platform for drug screening
Impact We have jointly identified a number of molecular processes linked to Parkinson's and other neurodegenerative disorders
Start Year 2007