MS04-P05 Protein misfolding in chimeras of the sh3 domain of the c-src and fyn tyrosine kinaseIn the next few years, due to the increase in life expectancy, neurodegenerative diseases, as for example the Alzheimer's disease, will be one major health problem in the developed countries. This disease belongs to the group of those caused by protein misfolding associated with the development of amyloid deposit. The study of the molecular basis of the missfolding processes is complicate due to the difficulties to obtain structural information at atomic level of such class of oligomers. However some proteins suffer misfolding forming oligomers which are able to crystallize. This oligomerization process is known as 3D domain-swapping (3D-DS), and the interchage of secondary structure elements may result in the formation of dimers, trimers, etc1. A case of study is the SH3 domain of the c-Src tyrosine kinase which develop intertwined dimers and, also, is able to form amyloid fibers. The formation of these 3D-DS structures and amyloids is pH dependent and very responsive to some puntual mutations2. Considering the results obtained in previous studies, our group have clonned, expressed and purified several chimeric proteins of the SH3 domain of the c-Src and Fyn tyrosine kinase, where the RT and n-Src loops of each domain have been interchanged3. In the c-Src SH3 the n-Src loop acts as hinge loop, which facilitate the opening of the domain to form the intertwined oligomer. We have study the stability of these proteins vs the pH (pH range 1-14) and in presence of the chemical denaturant guanidinium hydrochloride. These chimeric proteins are stable in a broad range of pH (pH 5.0-11.0) and show a high stability. The stability is higher at neutral pH than at acidic pH values. We have determined the oligomerization of the proteins by dynamic light scattering. The formation of dimers at acidic pHs is favoured by the addition of low molecular weight PEGs. Considering these results, we have screened the crystallization conditions at different pHs and in presence of several additives that promotes the formation of the dimers. Here we present the preliminary biophysical characterization of these chimeric proteins and their crystal structures determined at several pH values and oligomeric states. The results obtained show that besides residues at the hinge loops some residues al the distal loop might play a key role in the opening of the protomer to form the intertwined oligomers.
 Camara-Artigas, A. (2016). Arch Biochem Biophys, 602, 116-126.
 Bacarizo, J., et al. (2014) PLoS One. 9(12), 113-224.
 Camara-Artigas, A., et al. (2009). FEBS Lett, 583(4). 749-53.Keywords: SH3 domain, domain swapping, misfolding