The research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study.... Show moreThe research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study. Enzymes inherently embody a delicate balance between activity and stability, and the acquisition of new enzymatic functions is often accompanied by trade-offs, such as decreased stability or reduction of the original activity. Probing evolutionary adaptability of BlaC with laboratory evolution in combination with structural characterization can provide information about the mechanisms of rapid adaptations observed for β-lactamases in the clinic. The role of temperature as a conventional selection pressure in such evolutionary adaptation is unclear. The cooperative nature of enzyme unfolding over a narrow temperature trajectory raises the question whether evolution at temperatures well below the melting point is influenced by temperature. The approach used in this work to answer these questions is by simulating evolution under different selection pressures and characterize the variant enzymes in terms of activity, structure, dynamics and melting temperature. The research makes clear how enzyme kinetics and dynamics vary with different selection pressures and maps the evolutionary path that enzymes may take. The underlying structural mechanisms are established to provide a rationale for the observed effects. Show less
By utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and... Show moreBy utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and P450cam, while the results of PRE demonstrated the presence of a transient encounter complex. Furthermore, the significant insights of the interaction in the interface were uncovered by X-ray crystallography. Currently, the nature of Pdx effector activity is under debate. Since paramagnetic NMR experiments are applicable to solution studies at ambient temperature, PCS, RDC and PRE methods can further resolve the molecular mechanism of P450cam in the future. Show less
Receptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer.... Show moreReceptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer. The overexpression of EphA4, a member of the RTK family, has been observed in a variety of malignant carcinomas. The aim of the research project associated with this thesis was to develop high affinity inhibitors of the tyrosine kinase EphA4. Ligand discovery was based on two complementary approaches, a computational screen and an NMR based screen using Target Immobilized NMR Screening (TINS). In addition, orthogonal biophysical methods including Surface Plasmon Resonance (SPR) and protein observed NMR were employed to analyse fragment binding. The crystal structure of the EphA4 kinase domain was solved and the structure of the kinase domain in complex with dasatinib, a well-known kinase inhibitor, was also elucidated. The in silico approach discovered a potent inhibitor of EphA4 for which the binding mode was elucidated via X-ray crystallography. Moreover, the TINS approach identified two compounds that may constitute starting points for the generation of more potent EphA4 inhibitors. Show less
Verschillende biologische systemen, zoals het 50S ribosomale Hsp15 complex, een schistosomiasis diagnostisch antigen bindend antilichaam fragment (Fab 54-5C10-A), het zuurstof producerende enzym... Show moreVerschillende biologische systemen, zoals het 50S ribosomale Hsp15 complex, een schistosomiasis diagnostisch antigen bindend antilichaam fragment (Fab 54-5C10-A), het zuurstof producerende enzym chloriet dismutase (Cld) en het belangrijke regulatoreiwit van microtubule dynamiek (EB1) zijn onderzocht met geavanceerde biochemische technieken. De structurele informatie verkregen met electronenmicroscopie (EM), kleine hoek verstrooiing van R_ntgenstraling (SAXS) en R_ntgendiffractie aan individuele eiwitkristallen werd aangevuld met informatie uit andere biofysische en biochemische methoden, zoals kolom chromatografie, gel elektroforese, oppervlakte plasmon resonantie (SPR), massa spectrometrie (MS) en electron paramagnetische resonantie (EPR). Hoewel de in dit proefschrift beschreven biologische systemen uiteenlopen wat betreft chemische samenstelling, oorsprong en biologische functie, is er juist ook gekeken naar een gemeenschappelijk kenmerk: ge_nduceerde conformatieveranderingen binnen het eiwit, eiwit-RNA of eiwitcarbohydraat complex. Show less
In order to understand the function of a protein on a molecular level, the three-dimensional structure of the protein is often essential. X-ray crystallography is the primary method of protein... Show moreIn order to understand the function of a protein on a molecular level, the three-dimensional structure of the protein is often essential. X-ray crystallography is the primary method of protein structure determination. Despite recent rapid improvements in the field, the process of de novo structure determination may still take many months or years or may not be successful at all. The research described in this thesis is aimed at the improvement of the computational methods used for X-ray crystallography automated model building and refinement of macromolecular structures. A probabilistic approach is proposed in which a multivariate likelihood function that directly takes into account information from X-ray experiments is derived and shown to improve the process of protein model building and refinement. Show less
