Human ceruloplasmin and neurotransmitters: complex stabilization and crystallization

Dissertation to obtain a Master Degree in Molecular Genetics and Biomedicine at Faculty of Sciences and Technology,Universidade Nova de Lisboa

Human ceruloplasmin (hCp) is the molecular linker between the copper and iron metabolism and its importance in the homeostasis of human body has been implied in some neurological diseases. This plasma cuproenzyme has ferroxidase activity, oxidizing Fe2+ to Fe3+ and incorporating it into apotransferrin. hCp also has aminoxidase activity regulating the levels of amine stress hormones in the bloodstream and brain. Thus, it is thought to have an important role in neurodegenerative diseases such as Alzheimer’s or Parkinson’s. To know more about the role of cerulopalsmin on the oxidation of neurotransmitters and on brain homeostasis it is essential to know which protein residues are implied in the binding and stabilization of these neurotransmitters. The primary source of structural information for protein-ligand complexes is X-ray crystallography. This is the most successful method to determine macromolecular 3D structures but has some limitations as obtaining good diffracting protein crystals. In this study several attempts were made to achieve better hCp diffracting crystals and crystals of hcp in complex with dopamine, L-dopa, epinephrine or serotonin in order to further determine its tridimensional structure. To improve hCp stabilization and solubility, differential scanning fluorimetry and dynamic light scattering were used in a search for a better buffer for crystallization. For hCp crystallization the vapour-diffusion technique was used in combination with several other methods. Commercial crystallization screens, crystal seeding, additives, crosslinking were the several methods used to improve crystal diffraction. Co-crystallization of hCp with neurotransmitters was performed with no success. Soaking of hCp crystals with the neurotransmitters was performed in an attempt to get crystals of the hCpneurotransmitter complexes. All crystals were sent for analysis at European Synchrotron Radiation Facility (ESRF) and structural data will be further processed.