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ABSTRACT
Human interferon-gamma (hIFNγ) is a key cytokine in the realisation of cellular immunity. It accomplishes its biological activity upon binding to a specific cell receptor thus inducing the JAK/STAT1 signal transduction pathway. Two putative NLS sequences were pointed out to assist in the translocation of STAT1 into the nucleus. In order to employ mutational analysis for study the biological significance of the polybasic sequence Lys86-Lys87-Lys88 belonging to the upstream putative NLS, hIFNγ mutants with preserved structure and intact binding affinity to cell receptor need to be selected. To this end in silico studies of molecular stability of hIFNγ mutants was performed. The potential conformational changes in the structure of the mutant proteins were investigated employing molecular dynamics simulations. The free energy surface of Lys86 backbone torsion angles space in hIFNγ wild type and mutants was analyzed using metadynamic model. The obtained in silico results were verified by construction of selected mutant recombinant hIFNy proteins, which were analysed for biological activity. To judge for the secondary structure of the mutants the affinity to the cell receptor was investigated. High correlation between results of the molecular dynamics simulations and biological data was obtained.