Abstract
Cells rely on various biomolecular interactions and dynamics to perform their functions. The interacting building blocks range from small molecules to complex and dynamic molecular networks such as membrane-less organelles or the cell’s cytoskeleton. These systems play a significant role in biological function and the development of diseases. X-ray crystallography
... read more
and cryogenic-electron microscopy (cryo-EM) enable the atomic investigation of these complexes, by largely probing static states. As a result, insight into the role of dynamics, molecular subspecies or heterogeneity is often limited. Additionally, these structural biology techniques often rely on in-vitro sample preparations. Nuclear magnetic resonance (NMR) spectroscopy, however, can probe at dynamic systems under psychological conditions. Additionally, by utilizing solid-state NMR (ssNMR), it becomes possible to study complexes. This thesis presents a study of complex biomolecular systems by means of NMR. We focused on the system of microtubules (MTs) and their interactions with microtubule-associated proteins (MAPs) as they are essential to processes such as cell division, intracellular transport or cell structure. Moreover, we studied the gel-like phase of mRNA processing bodies (PBs) of S.Pombe which are involved in mRNA homeostasis. Chapter 2 presents the characterization of the microtubule-binding domain (MTBD) of microtubule-associated protein 7 (MAP7). NMR resonance assignment for this protein were obtained in solution and its secondary structure was characterized as an alpha-helical fold. In Chapter 3 we provided a protocol to isotopically label MTs for ssNMR experiments. A novel through-bond hCCH pulse sequence to study complex biomolecular systems was discussed in Chapter 4. This sequence is applicable to High-resolution Magic-Angle Spinning ssNMR and uses proton detection. We applied the sequence to labelled Tau K32 in complex with MTs and the fungal cell wall of S. commune. Building on these results, we examined in Chapter 5 the interaction of MAP7s MTBD with MTs. Furthermore, we combined this approach with cryo-EM and confirmed our observations with fluorescence anisotropy and solution-state NMR experiments. Combination of these results allowed us to obtain atomic information on the binding of MAP7, which suggests that it forms an elongated alpha-helix upon binding to MTs. By designing peptides compromising tubulin carboxy-terminal tails (CTTs), we also studied their interaction with MAP7 by solution-state NMR. A similar approach was employed to study the Tau-MTs interaction in Chapter 6. SsNMR was used to probe the rigid and flexible regions of the complex. Thus, we gained information about the Tau binding register that allowed us to extend a model in which Tau interacts with MTs. Moreover, we investigated the tubulin CTTs interaction with the protein, which led to the identification of involved residues in both interaction partners. In Chapter 7, we explored another complex molecular system. We conducted ssNMR experiments to investigate PBs. Our data shows that in a gel-like state, the overall protein conformations of the enhancer of decapping 3 (Edc3) and the decapping protein 2 (Dcp2) are conserved. We were also able to study dynamic changes upon protein interactions in this state and found that Dcp2 might persist in a dynamic equilibrium of conformations.
show less