Blanc SIMI 7 - Blanc - SIMI 7 - Chimie moléculaire, organique, de coordination, catalyse et chimie biologique

The timescales of disorder in proteins – DYN-IDP

Timescales of Disorder in Proteins

Intrinsically disordered proteins explored with nuclear spin relaxation

Timescales of Disorder in Proteins: a Methodological and Instrumental Approach

It is clear that many proteins execute essential biological functions with no well-defined 3D-structure. This is in direct contradiction to Pauling’s rule, which states that a functional protein should necessarily have a well-defined three-dimensional fold. Intrinsically disordered proteins (IDPs) constitute an important share of the eukaryotic proteome. Here, we use nuclear magnetic resonance (NMR) in synergy with other biophysical techniques to understand the conformational determinants of the function of disordered regions of the protein Engrailed 2 and the typical timescales of the exploration of this conformational space. <br />The study of the correlation times of motions in proteins in the nanosecond range requires the development of new methodological and instrumental tools. In particular, we have explored a new NMR approach: high-resolution relaxometry.

Nuclear magnetic resonance is a spectroscopic technique particularly suited for the characterization of disordered proteins. NMR provides quantitative and precise data with a residue or possibly atomic resolution. The exploration of motions on nanosecond timescales in proteins requires the development of new experimental approaches. We have constructed and used shuttle instruments, which permit the displacement of the NMR sample out of the magnetic center in order to explore magnetic-field dependent properties.
Besides, the study of the timescales of motions would be incomplete without a quantitative description of the conformational space of a protein and a characterization of the relationship between such physical and chemical properties and its functions. We have used a large number of NMR-based techniques in synergy with other biophysical approaches such as electron paramagnetic resonance and interaction studies with fluorescence anisotropy measurements.

We have assembled and adjusted a shuttle system designed to displace the NMR sample. This system benefit from a very high sensitivity. Besides, we have demonstrated the potential of high-resolution relaxometry on a model protein. We have also developed a new model for the interpretation of relaxation rates in disordered proteins. Last but not least, we have studied in detail the conformational space of the protein Engrailed 2 and the relationship with its function.
In order to pave the way for the future of high-resolution relaxometry, we have also designed theoretically a system to create multiple magnetic field plateaus in the stray field of a supraconducting magnet

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A first study of nanosecond motions has been pubished in 2013. Our new model for the interpretation of relaxation rates in disordered proteins was published in 2015 in Biophys. J. The design of magnetic field plateaus has been published in Sci. Rep. Sever

It has become evident that a large number of proteins fulfill essential biological functions, while lacking a well-defined three-dimensional structure. This is in direct contrast to Pauling’s rule, which states that a biologically active protein requires a native three-dimensional structure. Intrinsically disordered proteins (IDPs) constitute an important part of the eukaryotic proteome. The description of their conformational space is an active field of biophysical research. However, in striking contrast to folded proteins, description of their dynamics is nearly completely missing. Nevertheless, it is highly likely that dynamical behavior also play crucial roles in the biological functions of IDPs. Here, we introduce a variety of novel and powerful NMR relaxation-based approaches to sample the dynamics of IDPs.

Two different, yet complementary, NMR methodologies will be implemented. First, we will develop original and use existing NMR experiments to gather a complete set of information on the motions of the protein backbone of IDPs. This unprecedented set of experimental data on the dynamics of many internuclear backbone vectors and the correlation of these motions will be essential to understand the dynamics of unstructured proteins on a broad range of timescales. Second, we will develop new, innovative hardware for field-cycling studies of relaxation, which offers the combined advantages of high-resolution NMR spectroscopy and relaxometry.

This information will be collected on systems with important biological functions and that constitute potential therapeutical targets (Parkinson's disease, cancer). These proteins are: (i) two disordered natural partners of protein phosphatase 1, differing by their degree of residual structure, namely I-2 (high levels of residual structures), and spinophilin (low levels); (ii) Engrailed 2, a transcription factor that possesses a well-folded homeodomain and a long, mostly unstructured N-terminal domain. Thus, the investigation of these systems will cover a broad range of typical behaviors of IDPs that are currently described in the literature and thus provide much needed information. The newly gained insight of the dynamics at multiple timescales, as well as the detailed understanding of the nature of these motions will be essential to understand the mechanisms and function of IDPs.

Project coordinator

Monsieur Fabien Ferrage (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B) – Fabien.Ferrage@ens.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
CEA COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES - CENTRE DE FONTENAY-AUX-ROSES

Help of the ANR 200,000 euros
Beginning and duration of the scientific project: December 2011 - 42 Months

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