Equilibrium and kinetic folding studies of two YchN-like proteins: the Tm0979 dimer and the Mth1491 trimer

Proper folding of a protein to its native state is critical for the protein to be fully functional under biological conditions. Understanding protein folding and protein folding evolution within the same structural family are key to understand which processes assist or hinder protein folding and how to prevent misfolding. Tm0979 from Thermotoga maritima, Mth1491 from Methanobacterium thermoautotrophicum and YchN from Escherichia coli belong to the homologous superfamily of YchN-like proteins (SCOP and CATH: 3.40.1260.10). The structures of these proteins have been solved as part of structural proteomics projects, which consist of solving protein structures on a genome wide scale. In solution, Tm0979 forms a homodimer whereas Mth1491 folds as a trimer and YchN is a homohexamer. The structures of the individual monomeric subunits of these three proteins have high structural similarity, despite very low sequence similarity. The biological roles of these proteins are not yet well defined, but seem to be involved in catalysis of sulphur redox reactions. This thesis focuses on characterisation of the Tm0979 homodimer and the Mth1491 homotrimer, as well as the determination of the folding mechanisms of these two proteins. The folding mechanisms of the proteins are compared to each other and to the mechanisms of other dimeric and trimeric proteins. The evolution and basis of oligomeric structure within the YchN family are analyzed. Mutations of Tm0979 and Mth1491 are designed as a basis for future work to investigate processes responsible for switches in oligomeric protein quaternary.structure

Direct observation of heterogeneous amyloid fibril growth kinetics via two-color super-resolution microscopy.

The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson's disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures

The inverted free energy landscape of an intrinsically disordered peptide by simulations and experiments OPEN

The free energy landscape theory has been very successful in rationalizing the folding behaviour of globular proteins, as this representation provides intuitive information on the number of states involved in the folding process, their populations and pathways of interconversion. We extend here this formalism to the case of the Aβ40 peptide, a 40-residue intrinsically disordered protein fragment associated with Alzheimer's disease. By using an advanced sampling technique that enables free energy calculations to reach convergence also in the case of highly disordered states of proteins, we provide a precise structural characterization of the free energy landscape of this peptide. We find that such landscape has inverted features with respect to those typical of folded proteins. While the global free energy minimum consists of highly disordered structures, higher free energy regions correspond to a large variety of transiently structured conformations with secondary structure elements arranged in several different manners, and are not separated from each other by sizeable free energy barriers. From this peculiar structure of the free energy landscape we predict that this peptide should become more structured and not only more compact, with increasing temperatures, and we show that this is the case through a series of biophysical measurements. The free energy landscape of a protein provides a direct representation of the probability of measuring particular values of specific parameters that describe its conformational properties. The knowledge of the free energy landscape of a protein offers therefore the possibility of rationalising important aspects of its behaviour, including its stability, mechanisms of folding and molecular recognition, and the possibility of misfolding and aggregatio

Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell.

Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a "druggable" target with small molecules.The work was supported by a grant from the Development Gap Fund (MRC Technology), a research grant from the Isaac Newton Trust, Cambridge and from the CORE charity. LSI acknowledges the support of a Senior Fellowship from the Medical Research Foundation. TR holds a Royal Society University Research Fellowship. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ ERC grant agreement n° [279337/DOS]

Targeting the Intrinsically Disordered Structural Ensemble of a-Synuclein by Small Molecules as a Potential Therapeutic Strategy for Parkinson's Disease

Abstract The misfolding of intrinsically disordered proteins such as a-synuclein, tau and the Ab peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets a-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of a-synuclein-mediated dysfunction, including rescue of a-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of a-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting a-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions

Direct observation of heterogeneous amyloid fibril growth kinetics via two-color super-resolution microscopy.

The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson's disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures

Solution conditions determine the relative importance of nucleation and growth processes in alpha-synuclein aggregation

The formation of amyloid fibrils by the intrinsically disordered protein alpha-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that alpha-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of alpha-synuclein aggregation and aggregate spreading in the context of Parkinson disease

Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering

The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid β, can interact with lipids, and this interaction can strongly favor the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to vesicles formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the lipid vesicles do not simply act as a surface that catalyses the nucleation reaction, but that lipid molecules take an active role in the reaction. The binding of α-synuclein to the lipid vesicles immediately induces a major structural change in the lipid assembly, which leads to a break-up into small, cylindrical and disc-like lipid-protein particles. This transition can be largely reversed by temperature changes or proteolytic protein removal. Incubation of these small, cylindrical and disc-like lipid-α-synuclein particles for several hours, however, yields amyloid fibril formation, whereby the lipids are incorporated into the fibrils

N-Terminal Acetylation of α-Synuclein Slows down Its Aggregation Process and Alters the Morphology of the Resulting Aggregates.

Parkinson's disease is associated with the aberrant aggregation of α-synuclein. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and slows down both elongation and fibril-catalyzed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein exhibit a lower β-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behavior, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates