| dc.description.abstracteng | Intrinsically disordered proteins (IDPs) are proteins which lack a well-defined three- dimensional structure. The abundance and functional significance of IDPs has been recognized only recently. Due to their properties, IDPs play an important role in cellular functions. They serve as flexible inter-protein linkers, and participate in molecular recognition, molecular assembly, cellular signaling and regulation, or protein modification. Thus, genetically encoded alterations of IDPs are involved in many diseases, such as cancer, cardiovascular disease, amyloidosis, or neurodegeneration. Therefore the study and characterization of the conformational dynamics of IDPs are important to better understand the underlying mechanisms which lead to various pathologies.
FG repeats, rich in phenylalanine (F) and glycine (G), are one particular type of IDPs. FG repeats are located in the central channel of the nuclear pore complex (NPC), and they control the molecular transport between the nucleus and the cytoplasm. The way how FG repeats form or/and function as highly selective barriers in NPCs is not clear. In this thesis, the conformational dynamics of one FG repeats, Nsp1, is investigated by photo-induced electron-transfer fluorescence correlation spectroscopy (PET-FCS) and molecular dynamics simulation (MD simulation). Combination of PET-FCS and MD simulation offers a more comprehensive understanding of the relationship between functional mechanism and conformational dynamics of IDPs.
The results from PET-FCS measurements indicate that the N-terminus of Nsp1 tends to be more flexible than the C-terminus. Furthermore, short Nsp1 fragments (up to 50 amino acids) at low concentration (100 μM) do not tend to aggregate under physiological condition. These data indicate that the interaction between short FG repeats is not strong enough to solely generate the barrier. Interaction between long FG repeats or between many different kinds of FG repeats may also make a distinct contribution to the highly selective barrier of the NPC. To support MD simulations, hydrodynamic radii of various IDPs, FG repeats, GS repeats, Prothymosin alpha, and Integrase were measured by dual-focus fluorescence correlation spectroscopy (2fFCS). The data showed that the conformations obtained by the force field CHARMM 22* and a charm-modified TIP3P water model agrees best with the experimental data. These results are important for further force field developments of MD simulation for IDPs in the future. | de |