Caratterizzazione biochimica, biofisica e strutturale di RIM-BP in D. melanogaster

What allows to adapt organisms to rapidly changing events it's neurons ability to receive, integrate, store and send information to other neurons or tissues by means of electric coupling or chemical transmission. This last in particular involves the use of synaptic vesicles containing neurotransmitters which are released and restored at the presynaptic plasma membrane known as active zone (AZ). The complex exocytic-endocytic coupling of synaptic vesicles is coordinated though the interaction of two main complexes. The first is represented by proteins of the cytoplasmic matrix of the active zone (CAZ) which interacts with the second protein complex SNARE (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein (SNAP) receptor), located on the membrane of synaptic vesicles. The whole process of vesicles release is triggered by a starting depolarization which leads to the opening of Ca2+ voltage-gate selective channels and to further protein-protein interactions. One of the main protagonists involved in that coupling is the RIM-Binding protein (RIM-BP). This protein directly interacts with α1D (L-type) Ca2+ voltage-gate channel subunits and with the CAZ protein RIM, creating a tripartite complex responsible of Ca2+ channels regulation. Furthermore it shows an interaction between its first SH3 domain and the ELKS protein, better known in D. melanogaster as Bruchpilot, and between the two last SH3 domains and the three central fibronectin (FN3) domains within the same protein. Several other functions are supposed for RIM-BP, but its detailed function in the AZ remains to be determined. In order to learn more about RIM-Binding protein functions in this protein network, different domains from D. melanogaster were first purified and then investigated by means of X-ray crystallography or alternative techniques, such as mass spectrometry and CD spectra analysis. Co-crystallization of the SH3 II-III domains with the three FN3 allowed to obtain crystals several times improved, where the two SH3 domains alone did not yield to considerable results. The success of the complex crystallization can represent a confirmation of the expected interaction as determined by complementary methods. Two constructs of the N-terminal part of drRIM-BP were then purified and expressed. The poor results from crystallization attempts led us to employ the CD spectroscopy to investigate on secondary structures. By this technique we could show their temperature stability confirming that they are folded domains, and we could demonstrate that the N-terminal has a purely α-helical composition. We finally successfully carried out the cloning protocol for the first SH3 I domain with the goal of allowing future interaction studies with the CAZ protein Bruchpilot.