NMR study of protein dynamic changes upon ligand binding

Macromolecular interactions govern cellular functions. For the last decades, a multidisciplinary effort has been deployed to understand and characterize these interactions. Addressing these questions is especially important in order to design specific drugs in a therapeutic context. For this achievement, knowledge of the three-dimensional structure of molecular complexes is critical. That`s why a great structural work, by both X-ray crystallography and Nuclear Magnetic Resonance spectroscopy (NMR), has been deployed in order to solve the structure of molecular complexes. Nevertheless, the information provided by structural studies is necessarily incomplete without an accurate description of molecular dynamics. Indeed, protein dynamics are critical for biological function. In the case of macromolecular interactions conformational exchange, might contribute to binding properties and specificity. Unfortunately, in most cases, contribution of these motions to binding properties is poorly understood. Then, investigating dynamics involved in macromolecular interactions is of a major interest in order to better understand the molecular mechanisms critical for binding processes. In that context, the aim of this project is to investigate by NMR the protein dynamic changes occurring upon ligand binding. We will especially focus on understanding binding properties and specificity in the light of dynamic data. In particular, we are interested into the contribution of side-chains slow exchange motions (in the micro to millisecond timescale), which have never been taken into account for describing binding properties, whereas several evidences point out the role played by conformational exchange in binding processes. For this purpose, we will combine relaxation dispersion experiments with site specific labeling (via an original precursor synthetic approach) to study the motions on this particular timescale with an atomic resolution. We will also characterize the thermodynamic parameters of the complex formation via Isothermal Titration Calorimetry, and correlate these parameters to protein dynamics. As a model for this study we choose lipocalin Q83 in complex with enterobactin. This system give the possibility to label the ligand, allowing the measurement of dynamic changes for both partners and the characterization of the system with a precision and completeness never reach so far. Beside the fact that we would like to answer to fundamental questions such as why biomolecules interact in a specific but versatile manner, we would like to highlight the importance of the dynamic considerations in the understanding of binding processes. Indeed, so far, only static pictures (3D structures) are taken into account in the understanding of these processes and, more important, in drug design strategies. Whereas drug design approaches taking into account dynamic considerations should allow more efficient therapeutic interventions. We also believe that this study will demonstrate the necessity to provide an accurate description of molecular dynamics involved in biological processes and will inspire further similar studies on other relevant biological systems.