High troughput expression characterization and interaction studies of EF - hand calcium binding proteins (members of S100 family) (Doctoral thesis)

Καραβέλας, Τηλέμαχος Σ.


Calcium is known to play an important role in many biological processes. Beyond its structural role in biology it may be argued that it plays a significant role in solution, as well. A large variety of calcium-binding proteins, interact with calcium ions mediating important cell processes. For example, calcium may initiate the binding of several proteins to biological membranes or even act as an in-cell messenger. S100 protein family, one of the largest categories of calcium-binding proteins, consists of 20 protein members. S100 is a multigenic family of non-ubiquitous Ca2+-modulated proteins implicated in intracellular and extracellular regulatory activities, such as the conduction and transmission of the nerve impulse, muscle contraction, cell motility, cell growth and differentiation, gene expression and secretion. In addition, it has been reported that several human diseases, such as heart disease, neurodegenerative diseases, inflammatory disorders and cancer are closely connected to S100 proteins. In view of the importance of S100 in the cell physiology and moreover in eukaryotes, we selected to study two protein member of the family, S100A5 and S100A7. Initially, human S100A5 was cloned and expressed by bacterial cells (E.coli). Following the isolation and purification of the protein, CD and multidimensional NMR experiments were performed. CD studies of the apo and Ca2S100A5, revealed that the protein conserves its secondary structure, whereas thermal stability experiments were performed for both forms of S100A5 (apo and Ca-loaded). On the other hand, the homodimeric solution structures of S100A5 in both the apo and the calcium(II) loaded forms have been obtained, by means of NMR spectroscopy. S100A5 is a calcium binding protein of the S100 family, with one canonical and one pseudo EF-hand motif per monomer. It can bind four calcium ions, while it seems to bind one zinc ion per monomer. The structural differences resulting upon calcium binding change the global shape and the distribution of hydrophobic and charged residues of the S100A5 homodimer. This rearrangement involves, in particular, the hinge loop connecting the N-terminal and the C-terminal EF-hand domains, a reorientation of helix III with respect to helix IV, as common to several S100 proteins, and a longer alpha helical structure of helix IV. Relaxation data show a quite large mobility in the hinge loop which is not quenched in the calcium form. All these structural changes may be important for protein function. Furthermore, human S100A7 was cloned and expressed by bacterial cells (E.coli). Following the extraction from the cells and final purification of the protein, interaction studies, by means of NMR spectroscopy, with its possible biological cofactors, EFABP and S100A10, were performed. Interestingly, apoS100A7 interacts with EFABP, in solution, but several difficulties on spectra recording made difficult the complete identification of protein-protein interactions resulting in the solution structure of the complex S100A7/EFABP. On the other hand, apoS100A7 clearly shows distinct interactions with apoS100A10, a possible target protein revealed recently. This may implicate the dissociation of S100A7 homodimer and formation of the S100A7/S100A10 heterodimer. We generally believe, any available information, including all the above, concerning the structure, the individual functions and interaction with other target proteins, for these S100 members can comprise great tools in clinical diagnosis and/or treatment of S100 related diseases.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων Σχολή Θετικών Επιστημών Τμήμα Χημείας
Keywords: Πρωτεϊνες S100,S100A5,S100A7,Πυρηνικός μαγνητικός συντονισμός,Ιόντα ασβεστίου,Έκφραση ανασυνδυασμένων πρωτεϊνών,Δομή πρωτεϊνων,Δυναμική πρωτεϊνικών μορίων
URI: http://olympias.lib.uoi.gr/jspui/handle/123456789/600
Link: http://thesis.ekt.gr/thesisBookReader/id/28128#page/1/mode/2up
Appears in Collections:Διδακτορικές Διατριβές

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