Disordered states of proteins include the biologically functional intrinsically disordered proteins and the unfolded states of folded proteins which are important for protein folding and stability. Just as solving the structures of folded proteins has been extremely valuable in understanding their functions and properties, obtaining a comprehensive understanding of the structural characteristics of disordered states at a molecular level is crucial. The focus of this thesis is on combining experimental data with computational methods in order to probe the structural characteristics of disordered states at a molecular level. I developed a new method that combines different chemical shifts into a single residue-specific secondary structure propensity (SSP) score which I used to compare fractional secondary structure in alpha- and gamma-synuclein. Significant differences between the two suggested that gamma-synuclein might be protected from fibrillation due to increased helical propensity. I also introduced a new method for calculating residual dipolar couplings (RDCs) from disordered state ensembles by calculating local alignment tensors for short protein fragments. Using this method, I was able to predict experimental RDCs from statistical coil models containing far fewer structures than when global alignment is used, demonstrating that RDCs in disordered proteins are primarily determined by local structure. Finally, I made major improvements to the ENSEMBLE program which is used for calculating structural models of disordered states. I utilized large amounts of experimental data in order to calculate ensemble models of the Drosophila drkN SH3 domain unfolded state. Although highly heterogeneous and having broad molecular size distributions, the calculated ensembles have very different properties than expected for random or statistical coils and possess significant non-native alpha-helical structure and both native-like and non-native tertiary structure. This has significant implications for our understanding of the structural properties of protein disordered states in general.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/24365 |
| Date | 21 April 2010 |
| Creators | Marsh, Joseph Arthur |
| Contributors | Forman-Kay, Julie Deborah |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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