Browsing by Subject "intrinsically disordered proteins"

Intrinsically disordered proteins (IDPs) consist of charged, and polar amino acids, lacking bulky hydrophobic residues and they do not have a single well-defined 3D structure. They are found in all domains of life with higher abundance in eukaryotes, covering approximately 30% of the eukaryotic proteome. IDPs have key roles in many biological processes from cell signaling to phase-separation phenomena. Particularly, disordered protein regions serving as linkers, have been found in many multidomain proteins and they play a decisive role in the protein’s function. In the present thesis we aim to identify the correlation between sequence and rigidity disordered linkers, utilizing a synergistic method of Nuclear Magnetic Resonance (NMR) experiments and Molecular Dynamic (MD) simulations. For that purpose, glycine and proline rich disordered linkers which are widely utilized for constructing fusion proteins were used. Additionally, we aim to characterize the rigidity of the the disordered repetitive domain of the major ampullate type I dragline silk protein, using the same approach which served to connect the two terminal folded domains inside the protein. Dragline silk has been under thorough investigation due to its favorable mechanical properties and applications in material science. NMR spin relaxation times T1, T2 and hetNOE, are highly sensitive probes to motional timescales of IDPs, but they are difficult to interpret in terms of molecular dynamics. Here, we use the spin relaxation times to validate the MD simulations which in turn are set to interpret the linkers’ internal motions. Using the quality evaluation approach QEBSS, the best simulations were identified as the best description of the conformational ensemble, based on the comparison with the experimental spin relaxation times. Systematic differences in spin relaxation times correlate with systematic changes in the linkers rigidity, proving that spin relaxation times can be used to detect disordered linker rigidity. Prolines are shown to induce a comparatively expanded conformation ensemble with significantly slower dynamics whereas glycines offer flexibility. The ensemble of the repetitive domain of the silk protein showed conformations with intermediate rigidity. We also demonstrate that the synergy of NMR and MD simulations can be used for characterizing the rigidity-sequence interplay in short glycine and proline disordered linkers and silk protein systems. Being able to tune the properties of flexible and rigid linkers can be fundamental for understanding different biological systems and for protein engineering purposes. Bioengineering applications include designing and optimizing fusion protein linkers that in the long term be useful for drug design and developing protein-based biomaterials.