Comparison between importins-alpha of families alpha1 and alpha2 through structural and calorimetric technics

Abstract

The communication between the nucleus and the cytoplasm occurs through transport mechanisms that allow the passage of molecules through pores present in the nuclear envelope. Among the known transport pathways that enable the transport of macromolecules into or out of the nucleus through recognition of specific signal sequences, the so-called Classical Nuclear import Pathway is the most well characterized. In this way, the Importin-± protein (Imp±) identifies the proteins to be transported to the nucleus from the recognition of nuclear localization sequence (NLS). The structure of Imp± protein has been elucidated and characterized in some organisms. Through the analysis of amino acid sequences of these proteins, it was possible to classify them into three subfamilies: ±1, ±2, and ±3. The differences between the Imp± proteins from each family demonstrate the specificity in the recognition of NLSs, depending on the organism and function they perform. That is, an NLS peptide can even vary the affinity and the binding mode when interacting with distinct Imp± families. The objective of this project is to compare the affinity and the binding mode of the same NLS peptide with Imp± from ±1 and ±2 families. The NLSs used in this project will be of transcription factors of the fungus Neurospora crassa and the results of this study can contribute to the identification of specific characteristics of NLSs interaction with Imp± of Neurospora crassa. Initially, will be analyzed the affinity of the same fungus NLS with Imp± of Neurospora crassa (±1 family) and Mus musculus (±2 family). Then, the complex Imp±Nc / NLS peptide and Imp±Mm / NLS peptides will be tested by crystallisation experiments and the best crystals subjected to X-ray diffraction, collection and processing of data. After the modeling and refinement steps, the structures will be elucidated and compared to verify specificities, at structural level, of the interaction of the sam NLS peptide with Imp± from different families. The Structural Molecular Biophysics Laboratory of the Biosciences Institute of Botucatu, has the necessary infrastructure and personnel capable of performing all the experiments described in the project. This project is linked to the current thematic project approved by FAPESP (Proc. 2013 / 24705-3).