Although life is intrinsically related to biochemical and structural changes over time, the focus of structural biology has always been the detailed investigation of the highly populated compact ground states of biological macromolecules, yielding a static picture, and neglecting the dynamic nature of biology at the molecular level. At ambient temperature, proteins sample an ensemble of conformations as a result of their thermal energy. It is well known that states that are only sparsely and transiently populated can play critical roles in biochemical processes such as ligand binding, enzyme catalysis, protein misfolding, and amyloid formation. The multiplicity of states that are accessible to a protein is best described by a multidimensional energy landscape that describes the relative probabilities of the conformational states (thermodynamics) and the energy barriers between them (kinetics). The energy landscape not only depends on the particular protein but also on its environment. The interior of a cell, of course, is an extremely complex and crowded environment in which proteins and other macromolecules are present at concentrations of 300-400 mg/ml. The protein energy landscape also provides a description of the various conformtions that are sampled by a protein in the process of folding. Depending on the particular protein, there may be intermediate states represented as subsidiary minima on the energy landscape, and there may be more than one pathway for a protein to fold to its native state conformation. The folding problem of how proteins find their unique native states simply from the information contained within their amino-acid sequence remains a central unresolved question of modern science. Closely related to the protein folding problem is the question in which way proteins or protein fragments self-assemble into ordered functional macromolecular complexes, or insoluble aggregates associated with amyloid disease. It is generally believed that precursors of fibril formation are not the compact native protein states, but partially unfolded excited states that are populated at low level under native physiological conditions. Among the techniques nowadays available for detailed structure investigation, liquid-state NMR has the particular advantage of allowing the study of molecular structure in native-like solution conditions, cellular extracts, or even living cells. Multidimensional NMR is also ideally suited to access molecular dynamics and kinetics occurring over a wide range of time scales from picoseconds to seconds, hours and days, together with atomic resolution information for excited protein-state conformations. In addition, NMR enables atomistic descriptions of highly flexible conformational ensembles inter-converting on a sub-milliseconds time scale, as commonly encountered in natively unstructured protein fragments or partially denatured protein states. This versatility makes liquid-state NMR a unique tool for studying the 'invisible' manifold of high-energy excited molecular states, as well as conformational preferences in otherwise unstructured molecules. The research project proposed here aims at combining and further developing multidimensional NMR methods to study the structural, dynamic, and thermodynamic features of low-populated excited or transient intermediate protein states of different protein systems implicated in various biological functions. The high-resolution NMR studies will be complemented by low-resolution spectroscopic methods: fluorescence, circular dichroism, absorbance, and infrared correlation spectroscopy. These low-resolution techniques provide additional kinetic information of high precision for one or a few sites in the molecule, or an average of the entire system. The combined interpretation of different spectroscopic data shall provide a more comprehensive picture of the energy landscapes of these proteins, and thus a better understanding of the mechanisms of molecular recognition, protein folding and misfolding.
Monsieur Bernhard BRUTSCHER (Institut de biologie structurale)
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.
Institut de biologie structurale
Laboratoire de Chimie et Biologie des Métaux
Help of the ANR 308,866 euros
Beginning and duration of the scientific project: December 2007 - 36 Months