Jiggling and Wiggling Proteins Characterized by Solid-State NMR with Fast Magic Angle Spinning – FastSpinProts

In the past decades, solution-state Nuclear Magnetic Resonance (NMR) spectroscopy in liquids has evolved to become a powerful method for the study of internal mobility in proteins and other biomolecules, primarily through the measurement of spin relaxation rates and residual dipolar couplings. These allow one to probe internal motions occurring on different time scales, from picoseconds to milliseconds. Moreover, structural biology has demonstrated the fundamental role of the 3D structure of proteins to understand their biological function at a molecular level. In addition to this well documented aspect, recent studies have shown the importance of internal dynamics for molecular interactions. This adds a third dimension to the classical structure/function relationships, resulting in the more complex structure/dynamics/function relationship. Recent progress in solid-state NMR has opened the way to investigations of proteins in microcrystalline form. It has become possible to determine protein structures in the solid state. In addition, recent observations suggest that internal dynamics of microcrystalline proteins can also be accessed. In particular, nitrogen-15 spin relaxation rates have been measured in small proteins. This demonstrates the existence of motional processes on time scales that are significantly longer than those accessible by NMR relaxation measurements in solution. Several related objectives are pursued in this proposal, which converge to a common goal. One objective is the development of new approaches to the study of proteins in the solid state, based on promising methods that are currently under development. Another objective is the application of these new methodologies to the study of the internal dynamics of the human protein Centrin 2 (HsCen2) in micro-crystalline form. This proposal also aims at characterizing the internal dynamics in the complex formed by the C-terminal domain of this protein with a target peptide (P17-XPC). Such studies involve protein expression, purification and crystallisation, prior to the implementation of the solid-state NMR methodologies for probing internal dynamics. Finally, the model of Network of Coupled Rotators (NCR's) introduced by Partner 1 will be further developed and adapted to predict and interpret NMR relaxation parameters observed in solids. The enhancement of spectral sensitivity and resolution are of great importance. The use of radio-frequency (rf) irradiation with limited power is crucial to avoid damaging the samples, and may be achieved by combining fast spinning with specifically designed rf irradiation schemes. Fast spinning not only allows one to enhance both resolution and sensitivity but also has the advantage of removing contributions to relaxation due to spin diffusion between the abundant protons. However, fast spinning raises a number of methodological difficulties that we shall address, based on recent successful developments by Partner 1 that have allowed us to increase the efficiency of decoupling and to improve magnetization transfer by suitable methods for the recoupling of dipolar interactions. This will require the acquisition of dedicated instrumentation for an existing solid-state NMR spectrometer that will allow spinning of the sample at very high frequencies (on the order of 70 kHz). The understanding of dynamic properties of proteins is essential to be able to rationalize their function. This understanding is a precondition for rational drug design, which is obviously of major economic and societal importance. The present proposal is based on the complementarity of the skills of three partners covering the fields of NMR methodology, protein dynamics, and structural biology based on X-ray diffraction. The synergy between the partners brought together in this project will provide a better understanding of the dynamic behaviour of the human centrin protein, and produce new methods for future studies of a broad range of biomolecules.