Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / BRUTSCHER Bernhard

Protein & RNA Folding and Methods development


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Team members

Bernhard. Brutscher
Adrien Favier
Isabel Ayala
Lionel Imbert
Sergio Gil-Caballero
Zsofia Solyom

Intrinsic disorder in proteins: transient structure and molecular recognition elements

The structure-function paradigm stating that a well-defined structure is required for a protein’s function has been challenged by the discovery of a large number of highly flexible proteins or protein segments that exist as ensembles of partly collapsed or extended structural conformers in the cell and that are functional as such. Intrinsically disordered proteins (IDPs) or protein regions (IDRs) have been shown to play important roles in regulatory and signaling processes where the structural flexibility allows the protein to adapt to and interact with a large number of distinct molecular partners. Similarly, structural disorder is also abundant in viral proteins. Viruses are often characterized by a small genome, only coding for a few proteins. High mutation rates in these genomes allow to adapt to changing environments and to escape the defense mechanisms of the host cell. Again, structural flexibility presents a functional advantage in terms of binding promiscuity, as well as a high tolerance to mutations.
Currently, we investigate several viral IDPs, among those non-structural protein 5A (NS5A) from the hepatitis C virus. NS5A plays an important role in viral replication and particle formation. The C-terminal 250 residues are highly disordered, and display numerous interaction sites for other viral and host proteins. Our recent study revealed the presence of transient α-helical structures in 4 regions of the peptide sequence that are partly stabilized by long-range tertiary interactions. Two of these transient α-helical regions form a non-canonical binding motive for low affinity binding to SH3 domains.

Collaborations: D. Willbold (FZ Jülich, Germany). This project is part of the Marie-Curie Training Network (ITN) IDPbyNMR.

Real-Time NMR Investigation of Protein Folding and Transiently-Populated Intermediate States

Structural biology generally focuses on studies of the highly populated ground states (native states) of proteins. However, it has long been recognized that higher energy conformational states can play important functional roles, or may be responsible for protein misfolding and fibril formation leading to various human diseases. In our team we use off-equilibrium real-time multidimensional NMR spectroscopy to obtain detailed information on protein folding and aggregation pathways, and to investigate at atomic resolution the structure and dynamics of protein states that are separated from the ground state by energy barriers of > 10 kcal/mol. We are especially interested in the amyloidogenic human protein β2-microglobuline that is responsible for fibril formation in the joints and connective tissues of patients undergoing long-term dialysis.

Collaborations: A.Corazza, G. Esposito (Univ. of Udine, Italy); V. Forge (iRTSV, CEA Grenoble, France)

RNA conformational transitions and RNA chaperone activity

Many natural RNAs have been shown to form, in addition to their ground-state structure, alternative, metastable structures that are relevant for their function in the cell. Conformational transitions of RNA play fundamental roles in the processes of RNA refolding and annealing, in the catalysis of chemical reactions by ribozymes, and in the regulation of gene expression by riboswitches. Riboswitches are cis-acting regulatory elements found in the non-coding regions of several messenger RNAs. They modulate gene expression, without the assistance of proteins, in response to particular environmental conditions like nutrient availability. The regulation of gene expression depends on the conformational rearrangement of the riboswitch in response to direct binding of the nutrient in a specific pocket or aptamer in the RNA. The RNA refolding upon ligand binding can be conveniently studied by real-time multi-dimensional NMR spectroscopy.

In addition, we are interested in the mechanisms of RNA chaperone proteins that assist in RNA folding. RNA chaperone proteins typically bind RNA transiently and with low specificity. Much is known about how proteins bind RNAs specifically and many high-resolution structures of RNA-protein complexes are available. However, little is known about the interaction mode of proteins binding RNA weakly and transiently, such as RNA chaperones. Therefore it is of highest interest to investigate the functional and structural properties of these proteins in order to elucidate their mode of action. Currently we focus on 2 RNA chaperone proteins, the globular well folded bacterial CspA, and an intrinsically disordered viral core protein. The aim of this project is to come up with a comprehensive understanding of the atomistic details of this very important biological process.

Collaborations: C. Kreutz (Univ. of Innsbruck, Austria); R. Konrat (Univ. of Vienna, Austria)

NMR methods development

A major research theme of the team is the development of new pulse sequences and innovative spectral analysis tools for biomolecular applications. In recent years, we have particularly focused on fast multidimensional data acquisition techniques.
Multidimensional NMR offers the spectral resolution required to resolve the many resonance peaks of macromolecular systems by spreading them along multiple dimensions. However, multidimensional NMR requires long measurement times that are exponentially increasing with the dimensionality of the experiment. Fast acquisition techniques are required for investigation of molecular systems that are intrinsically unstable under physiological conditions, and of transient conformations accumulated during protein folding. They will also impact high-throughput NMR studies, such as the systematic evaluation of bio- and physico-chemical conditions under which the final structure investigation by either NMR or X ray crystallography will take place, or library-based ligand screening. Finally, the increased rate of NMR data collection provides new exciting opportunities for site-resolved kinetic NMR experiments, such as real-time studies of protein and RNA folding, conformational changes related to biomolecular function, or the measurement of fast hydrogen-deuterium exchange rates in partially unstructured native states or molten globular states of proteins. Examples of fast NMR tools developed in our laboratory are fast-pulsing NMR, projection NMR, Hadamard spectroscopy, optimized spectral aliasing, covariance-based methods, and non-linear data sampling and processing.