Development and application of novel NMR methodology for elucidation of protein structure and dynamics

The design of a novel drug is a creative act. The difference between a researcher and an artist, besides the pursuit of a particular goal, is that his/her creativity is based on scientific knowledge and technology. Until today, drug discovery was mainly dominated by trial and error, based on empirically derived rules. Today, the “trial and error” approach is being replaced by a conscious design based on improved predictions. These improvements in the prediction of the structure of a drug molecule are mainly based on the increasing knowledge about molecular mechanisms. [1] This increase of mechanistic knowledge is also caused by the progress of structure elucidation techniques like NMR-spectroscopy or X-ray crystallography. So far, our understanding of molecular mechanisms is based on the justified principle: Structure determines function. The number of deposited protein structures grows exponentially. Nevertheless, the ratio between solved and unsolved human protein structures is in the single-digit percentage range. [2] This relative number is even lower for proteins that are insoluble and at the same time amorphous in the solid-state, such as fibrils or membrane proteins. The structure of such proteins of great importance is not accessible by X-ray crystallography or liquid-state NMR-spectroscopy. In order to close this gap, a new NMR methodology has been developed over the last two decades, the proton-detected fast-magic-angle-spinning solid-state NMR-spectroscopy. This new method enables the structure-elucidation of such amorphous and insoluble proteins. In this context, Bernd Reif et al., Rasmus Linser et al., Guido Pintacuda et al., and others did the pioneering work. In this line, an important objective of this thesis was to contribute to the continuous development of this methodology. The key to well-resolved protein structure from NMR-spectroscopy is precise distance restraints. However, till date, solid state NMR-spectroscopy has only been able to provide qualitative restraints, grouping the internuclear distance as close, medium, or far. Opposed to the general picture, with the help of Suresh K. Vasa, Evgeny Nimerovsky, Himanshu Singh, Beat Vögeli, and others, I developed a user-friendly approach to determine accurate distance restraints in solid-state NMR. Hereby, all site-specific errors that occur during magnetization transfer are addressed by an integrated approach. The approximations to be made are carefully validated by numerical simulations. Further, in order to address challenging protein targets where the assignment might be ambiguous and incomplete, I have performed the first kinetic hydrogen-deuterium exchange measurements in solid-state NMR-spectroscopy in collaboration with Suresh K. Vasa, Himanshu Singh, and others. The method reports on the over-all positioning of exchangeable protons within the protein by information on the presence in the hydrophobic core or on the hydrophilic surface and on the presence and strength of structural hydrogen bonds. Therefore, this information can be used for assignment purposes and for structure determination. Hereby, it was crucial to be able to separate exchange hindrance due to hydrophobic shielding from the hindrance due to hydrogen bonding. Experimental site-specific information about hydrogen bond strength is valuable for determination of protein structure and dynamics simultaneously. In addition to a well-defined distance restraint, a hydrogen bond also reports on the rigidity of structural elements. In particular, the stabilization of functionally important loop tips by sidechain-to-backbone hydrogen bonds is investigated in this work. While the interplay of protein structure and function is well accepted and understood, the current state of understanding how local motion contribute to functional mechanisms leaves much to be discovered. In this context, NMR spectroscopy is one of the techniques of choice. Nowadays detailed information on directional protein motions is provided primarily by the theoretically based MD simulations. Another part of this work is dedicated to advance and apply NMR-spectroscopic methods, which have been developed to determine these directional dynamics and lead towards interpretations of the biological function. Along these lines, in liquid-state protein NMR, Vögeli et al. recently developed an approach that provides distance restraints with such accuracy that differences between distance restraints and average atomic positions are no longer associated with experimental error but with protein dynamics. With the help of Cornelia Hebrank, Snehal Patel, and Lars V. Schäfer, this method was applied to the protein binding domain SH3, yielding interesting mechanistic insights regarding ligand binding. For the first time, the novel method was evaluated using state-of-the-art MD simulations and further compared with the dynamics data using other NMR approaches. In addition to providing mechanistical insides for the protein SH3, the value of this new approach, which is the first experimental method for the determination of spatial dynamics, is emphasized. In general, the main objective of this work was to develop applicable methods for the benefit of other researchers and thus for the benefit of science and humanity