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.

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.

Master of Science / Department of Biochemistry / Jianhan Chen / Paul E. Smith / Intrinsically disordered proteins have been shown to be important in many physiological processes, including cell signaling, translation, and transcription. They are also associated with cancer, and neurodegenerative diseases. The tumor suppressor p53 contains several disordered regions, including the C-terminal negative regulatory domain (NRD). In cancer the function of p53 has been shown to be repressed by S100B(ββ) binding to p53-NRD. Binding of S100B(ββ) blocks acetylation and phosphorylation sites in the p53-NRD, which leads to tetramer dissociation and prevents p53 activation. NMR studies have shown that p53-NRD binds S100B(ββ) in a stable α-helix conformation. Interestingly, despite the well-converged and apparent rigid nature of the NMR structure ensemble, a majority of intermolecular NOEs used to calculate the NMR ensemble are very weak (≥6 Å). The final NMR structures also contains unsatisfied buried charged residues at the binding interface. It’s plausible that the p53-S100B(ββ) complex is more dynamic than previously believed. The goal of the study is to determine the potential conformational heterogeneity in p53-S100B(ββ) complex using molecular modeling. For this, five diverse structures were selected from the 40-member NMR ensemble. For each initial conformation, we performed 100 ns molecular dynamic simulations in explicit solvent to explore the structure and dynamics of the p53-NRD in complex with S100B(ββ). Several analytical tools were used to characterize the p53-NRD conformation, including root-mean squared deviation (RMSD), root-mean squared fluctuation (RMSF), and residue helicity. The accuracy of the simulations was mainly assessed by comparing with experimental NOEs. The results show that, even though the ensemble is heterogeneous it satisfies 82% of the experimental NOEs. Clustering analysis further suggests that many conformational sub-states coexist for this complex, and individual clusters appear to satisfy only subsets of NOE distances. Importantly, the buried surface analysis demonstrates that the heterogeneous ensemble generated from MD provides similar shielding of key residues, which include post-translational modification residues needed for p53 activation. This study also demonstrates that atomistic simulations can provide important insights into structure and dynamics of IDPs for understanding their biological function.

No description available.

Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2). Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions. Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18). / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

Research into the relationship between the structure and function of proteins has been ongoing now for several decades. More recently, there has been an explosion in the investigation of the dynamic properties of proteins, and how their dynamic propensity relates to their function. This new direction in protein research requires new techniques to analyse protein dynamics, since most traditional techniques are biased towards a fixed tertiary structure. Mass spectrometry (MS) is emerging as a powerful tool to probe protein dynamics since it can provide information on interconverting conformations and has no preference towards the folded state. Furthermore, its low sample consumption, rapid data acquisition and low data processing positions MS as an attractive tool in protein structure research. The hybrid technique of ion mobility-mass spectrometry provides further insight into the range of conformations adopted by proteins and protein complexes, by providing information on the size in terms of rotationally averaged collision cross section. The work presented in this thesis considers proteins with a range of structural characteristics. We use ion mobility mass spectrometry to investigate proteins of different extents of disorder, protein complexes with dynamic entities and a system that undergoes structural rearrangement upon ligand binding. First, a framework of mass spectrometry experiments is described which allows identification of the extent of structure and disorder within proteins. This framework is tested on a range of different systems throughout the thesis. Differences in the gas-phase properties of two conformationally dynamic proteins which behave similarly in solution are investigated and from this research we postulate a new ionisation mechanism for partially folded proteins. The dynamic propensity of C-terminal p27 is investigated and compared to two permutants which allows us to delineate how the location of charged residues in a primary sequence affects the structure of a protein. We monitor the 'folding-upon-binding' behaviour of p27 upon association with its binding partners, and how this differs with the order of charged residues in the linear sequence. Finally, we describe the structural rearrangement of Fdc1 upon the binding of its cofactor; a prenylated FMN molecule. This thesis demonstrates the suitability of ion mobility-mass spectrometry for the investigation of dynamic properties of proteins and protein complexes.

For half a century, the central tenet of protein science has been grounded on the idea that the three-dimensional structure of a protein underlies its function. However, increasing evidence of natively unstructured but functional proteins is accumulating. Termed as intrinsically disordered proteins (IDPs), they populate a number of different conformations in isolation. Interestingly, as part of their function, some IDPs become fully or partly structured upon interaction with their binding partners. This process, known as coupled folding and binding raises the question what comes first - folding of the IDP or binding to its partner protein followed by folding. This thesis focuses on understanding the role of disorder in protein- protein interactions using biophysical characterization. Over-representation of IDPs in complex network and signalling pathways emphasizes the importance of disorder. Conformational flexibility in IDPs facilitates post-translational modifications, which provides a neat way to modulate the residual structure. This can alter affinity of IDPs to their partners and it is speculated that bound like structures of IDPs speed association. The impact of phosphorylation was explored in the KID/KIX system: phosphorylation modulates only the dissociation kinetics increasing the lifetime of the bound complex, which may be important in signalling processes. Further, phi-value analysis applied to investigate the mechanism of interaction reveals that non-native interactions play a key role in this reaction, before the IDP consolidates its final structure in the bound complex. Promiscuous interaction of IDPs with their partners often results in complexes with differing affinities. Members of BCL-2 family were explored, and the results indicate that IDPs bind to the same partner protein with marginal variation in the association rates, but significant differences in dissociation rates are observed. Thus, it seems that in such homologous but competing network of proteins, disorder facilitates complexes with differing affinities by modulating dissociation rate, again altering the lifetime of the bound complex. The work presented here demonstrates that disorder plays a role in altering complex lifetimes. Perhaps being disordered permits a level of plasticity to IDPs to adapt the rates at which they bind/unbind to many target proteins. This may be why disorder is conserved and abundant in proteins involved in intricate signalling networks.

Conformational ensembles are models of proteins that capture variations in conformation that result from thermal fluctuations. Ensemble based models are important tools for studying Intrinsically Disordered Proteins (IDPs), which adopt a heterogeneous set of conformations in solution. In order to construct an ensemble that provides an accurate model for a protein, one must identify a set of conformations, and their relative stabilities, that agree with experimental data. Inferring the characteristics of an ensemble for an IDP is a problem plagued by degeneracy; that is, one can typically construct many different ensembles that agree with any given set of experimental measurements. In light of this problem, this thesis will introduce three tools for characterizing ensembles: (1) an algorithm for modeling ensembles that provides estimates for the uncertainty in the resulting model, (2) a fast algorithm for constructing ensembles for large or complex IDPs and (3) a measure of the degree of disorder in an ensemble. Our hypothesis is that a protein can be accurately modeled as an ensemble only when the degeneracy of the model is appropriately accounted for. We demonstrate these methods by constructing ensembles for K18 tau protein, \(\alpha\)-synuclein and amyloid beta - IDPs that are implicated in the pathogenesis of Alzheimer's and Parkinson's diseases.

Tat1-72, is an intrinsically disordered protein at pH 4.1 as previously indicated by NMR chemical shifts and coupling constants, and confirmed by 15N-relaxation parameters. The presence of SDS elicits a conformational change to α-helicity in Tat1-72. In the presence of the non-ionic DDM detergent and zinc, Tat was found to be soluble at pH 4 when bound to TAR RNA; TAR binding also elicits a conformational shift to α-helicity in Tat1-72. The β-sheet content of Tat1-72 is increased in the presence of NaCl. In similar conditions, Tat1-72 aggregates stained with Congo Red displayed a yellow-green birefringence and a red-shift in the Congo Red absorbance that is typical of β-amyloid fibril. The web-based algorithm “WALTZ” identifies the majority of the Tat1-72 hydrophobic core region as amyloidogenic. The helical propensity of Tat1-72 in TFE was determined by two-dimensional NMR spectroscopy.

Tat1-72, is an intrinsically disordered protein at pH 4.1 as previously indicated by NMR chemical shifts and coupling constants, and confirmed by 15N-relaxation parameters. The presence of SDS elicits a conformational change to α-helicity in Tat1-72. In the presence of the non-ionic DDM detergent and zinc, Tat was found to be soluble at pH 4 when bound to TAR RNA; TAR binding also elicits a conformational shift to α-helicity in Tat1-72. The β-sheet content of Tat1-72 is increased in the presence of NaCl. In similar conditions, Tat1-72 aggregates stained with Congo Red displayed a yellow-green birefringence and a red-shift in the Congo Red absorbance that is typical of β-amyloid fibril. The web-based algorithm “WALTZ” identifies the majority of the Tat1-72 hydrophobic core region as amyloidogenic. The helical propensity of Tat1-72 in TFE was determined by two-dimensional NMR spectroscopy.