Biological motion constitutes a key and indispensable element of all biomolecules, as dynamics tightly link spatial architecture with function. Several computational and experimental techniques have been developed to study biomolecular dynamics. Nevertheless, few label-free and atomic or sub-atomic resolution techniques are able to capture biological motions at close to native conditions. Indeed, the only label-free technique giving atomic level access to dynamics from picoseconds down to seconds is nuclear magnetic resonance (NMR) spectroscopy. In this dissertation, I identify the imperfections and inaccuracies accompanying the routine and well-accepted methods of probing protein dynamics via 15N spin relaxation NMR measurements. Subsequently, I propose and develop solutions and experimental approaches to overcome the limitations and eliminate artefacts. The routine procedures applying heavy water as an internal locking standard lead to artifacts in every type of relaxation rate of 15N amides due to reaction with exchangeable deuterons. The deviations from correct values are most pronounced for highly dynamic and exposed protein fragments. I introduce a novel set of directly detected 15N spin relaxation experiments yielding an unprecedent resolution resolving the signal overlap, although of lower sensitivity. I propose a more accurate.
Finally, I present how the 15N spin relaxation techniques and improved routines can be applied to understand biological processes that cannot be described without monitoring molecular motions. Using the example of human BTB domains, which are directly linked to human cancer, I demonstrate the ability to detect cryptic binding sites on the surfaces of proteins. The cryptic binding site was verified by a comprehensive NMR-monitored fragment-based screening that revealed a hit-rate only for MIZ1BTB, which was the only protein displaying slow segmental motions. I also managed to track subtle and biologically-relevant dynamic modulations of an exposed H3 histone tail affected by H1 histones or other histone variants. Enhancement of H3 tail dynamics led to increased H3K36 methylation, while restriction of motions resulted in the opposite effect. These observed correlations unequivocally support the essential role of molecular mobility in biological functions.
|Date of Award||Dec 2021|
|Original language||English (US)|
- Biological, Environmental Sciences and Engineering
|Supervisor||Lukasz Jaremko (Supervisor)|
- protein dynamics
- fragment-based screening
- pulse programming
- biological motions