Theory and Applications of Nuclear Magnetic Resonance of Biomolecules in solution and in the solid state - Teoria ed Applicazioni della Risonanza Magnetica Nucleare di Biomolecole in soluzione ed allo stato solido

This thesis describes the application of Nuclear Magnetic Resonance (NMR), with particular reference to paramagnetic effects, to the characterization of structural and dynamical features of some biomolecules. The research re- ported is framed into a critical conspectus of the state of the art of biomolec- ular NMR. Motions in solution give rise to sets of averaged experimental observables. The reconstruction of the motion involved in the averaging is not a triv- ial task: a method is developed to obtain accurate information on dynamics from average data. Paramagnetism-based restraints obtained in solution are used to study the dynamics of two-domain proteins calmodulin and matrix- metalloproteinase 1 (MMP1). In the solid state any molecule is surrounded by a number of neighbors: thus if the molecule is paramagnetic, it will sense a contribution to paramagnetic observables also from crystal neighbors. To recover this information an algo- rithm is developed and implemented. Such contribution is explicitly included in the calculations as restraints obtained in the solid state and allows for the refinement of the structure of MMP12 and of its crystalline environment at the same time. Solid state NMR is gaining interest because it allows one to overcome some limitations (especially in size) of solution state NMR, and has recently seen a profound development. Anyway, sample preparation in solid state NMR has not completely followed up: usually microcrystalline preparations are employed to achieve high resolution, yet protein crystallization is a highly unpredictable process and simpler approaches are sought for. We developed a new technique, that we refer to as “sedimented solutes NMR” (SedNMR), that allows for the observation in the solid state of soluble macromolecules without the need for crystallization, lyophilization or freezing. A theoretical description of the process is provided and, from this, a number of experiments are proposed.