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Molecular bases of specificity and affinity in partner recognition by intrinsically disordered proteins (IDPs)

Heads Sonia LONGHI

People involved: A. Gruet (AI, CDD, EPHE student), A. Vassena (Master2 student, Erasmus, Universita’ degli Studi Milano-Bicocca), M. Dosnon (Master2 student, BBSG, Aix-Marseille University).

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Figure 7
Schematic illustration of the split GFP re-assembly principle. The complex resulting from the re-assembly of the two GFP fragments (with the N-terminal and the C-terminal fragments of GFP being fused to either NTAIL or PXD, respectively), is fluorescent, contrary to the isolated fragments that are insoluble in E. coli. Re-assembly relies on the ability of the fused proteins (NTAIL and PXD) to interact with each other. The fluorescence is proportional to the affinity between NTAIL and PXD.

One of the most intriguing features of IDPs is their ability to recognize multiple partners while conserving specificity and often displaying a good affinity (nM-µM range). In order to unravel the determinants of specificity and affinity in partner recognition by IDPs, an in vitro evolution approach is currently being used to generate amino acid diversity within the C-terminal domain of the MeV nucleoprotein (NTAIL). A library of NTAIL mutants has been generated (collaboration with C. Bignon, AFMB) and screening of variants with altered binding properties was carried out based on split GFP re-assembly (Figure 7). Clones have been characterized in terms of their binding abilities towards MeV PXD and of their nucleotide sequence. The interaction between a set of NTAIL variants and the partner protein are currently being investigated using spectroscopic approaches and ITC. A few crystallization trials on rationally designed complexes endowed with less flexibility are in progress. The interaction between NTAIL variants and PXD is also being studied in silico using molecular dynamics simulation approaches (collaboration with P. Roche, IMR, Marseille). Finally, the functional impact of NTAIL substitutions is being assessed in vivo in the context of MeV infection (collaboration with D. Gerlier, Lab. Virologie Humaine, Lyon).

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