NMR Application In Fragment-Based Screening Against TDP-43 Tandem RRM And In Dynamics Of HADK2

This thesis contains two main parts.The first and third chapters present the NMR fragment-based screening against Tandem RNA Recognition Motifs(RRMs)of TDP-43,and the fourth and fifth chapters describe the application of NMR relaxation dispersion and Pseudocontact shifts methods for getting information about the ground-state to excited-states exchange in human ADK2(hADK2).In the midst,the second chapter describes the methods and materials used in this whole work.TDP-43 is originally a nuclear protein but translocates to cytoplasm in the pathological condition.TDP-43,as an RNA-binding protein,consists of two RRMs(RRM1 and RRM2).RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules.Thus,they assist the entire TDP-43 protein to participate in neurodegenerative and cancer diseases.Consequently,they are potential therapeutic targets.Protein-observed and ligand-observed nuclear magnetic resonance(NMR)spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43.We identified three hits weakly binding the tandem RRMs by using the ligand-observed NMR fragment-based screening.The binding topology of these hits is then depicted by chemical shift perturbations(CSP)of the 15N-labeled tandem RRM and RRM2,respectively,and modeled by the CSP-guided High Ambiguity Driven biomolecular DOCKing(HADDOCK).These hits mainly bind to the RRM2 domain,which suggests the druggability of RRM2 domain of TDP-43.These hits also facilitate further studies regarding the hit-to-lead evolution against TDP-43 RRM domain.Biological macromolecules are highly flexible and continually undergo conformational fluctuations on a broad spectrum of timescales.The focus of structural biology is mostly on studies of the highly populated,ground states of biological molecules.The states that are only sparsely and transiently populated are more difficult to probe because they are invisible to most structural methods.Yet,such states can play critical roles in biochemical processes such as ligand binding,enzyme catalysis,and protein folding.A description of these states in terms of structure and dynamics is,therefore,of great importance for understanding the protein functions.The advances in solution nuclear magnetic resonance(NMR)spectroscopy allowed the bacterial Adenylate Kinases dynamics to be observed with detail.However,nothing is known on conformational changes and dynamics of human Adenylate kinases.We have used the NMR relaxation dispersion(CPMG)to detect the hADK2 excited-state conformations exchanging with the ground state in the timescale of microsecond to millisecond.We also labeled hADK2 with lanthanide-chelating peptides,which paves way for the following experimental observation of Pseudocontact Shifts(PCSs)of excited-states in slow exchanging with the ground-state.