Comparative study between different biophysical techniques for further understanding of the nuclear importation mechanism

Abstract

The quantitative study of the protein-binding interaction has been shown to be essential for the study of nuclear import mechanisms. Particularly, in the so-called classical nuclear import pathway these data are essential, because import±-± (Imp±) is coupled to cargo proteins to be transported to the nucleus from nuclear localization sequence (NLS) recognition. Imp± are classified into different subfamilies depending on the organism and the proteins they transport. This specificity between different Imp± and NLSs of cargo proteins is a subject that is currently being researched by us and some other groups and has direct applications in the area of drug development. Currently, our research group is dedicated to the study of Imp± of several organisms, including that of the genus Leishmania and different human isoforms, which are the subject of two thematic projects of our team. Thus, the improvement in the use of biophysical methods of analysis of the interaction Imp± / NLS of the cargo proteins has a fundamental role and is the goal of this PhD project. The project has three main objectives: i) to compare the interaction of the Imp± with NLSs by different techniques, such as by isothermal titration calorimetry (ITC), fluorescence spectroscopy and microscale thermophoresis (MST), analyzing the main differences between each of them. This comparison is little discussed in the literature, but essential in scientific terms, since the results obtained by different techniques, usually present large variations; ii) comparative study between different NLSs and Imp± aiming a better understanding of the specificities of this interaction, using ITC and computational modeling; iii) development of an ITC data analysis algorithm, since commercial software presents difficulties to calculate parameters in more complex systems. For example, in the case which there are structurally different sites of binding in the protein by a ligand, such as Imp±/NLSs systems. The project has great scientific and technological relevance and presents a high degree of innovation, since it can bring advances in the study of mechanisms of nuclear proteins transport in several organisms and will allow an important development in terms of the use of biophysical techniques to characterize protein-ligand for several biological systems. Studies with Imp± from different organisms are being carried out in collaboration with Prof. Maria Isabel Nogueira Cano (IBB/UNESP, Botucatu) and directly related to the recently approved FAPESP (proc. 2018/04375-2) thematic project that has Prof. Cano as coordinator and Prof. Fontes as official collaborator. This study is also a fundamental part of the thematic project submitted to FAPESP (proc. 2019/03108-3), which is coordinated by Prof. Fontes. (AU)