Molecular Dynamics Simulations of His$$_6$$-FLAG-hIFN$$\gamma $$ Fusion Glycoproteins

Human interferon-gamma (hIFN\(\gamma \)) is a key immunomodulating secretory glycoprotein. Although glycosylation is not necessary for its activity, it does affect the physico-chemical properties of the cytokine. Recently, a technology was developed for the secretory expression of glycosylated hIFN\(\gamma \) and its highly prone to aggregation mutant K88Q in insect cells. In addition, the proteins were labeled with specific tag peptides added to their N-termini. It was experimentally observed, that the obtained fusion proteins have significantly reduced biological activity and when glycosylated, they were resistent to enterokinase action and the tag could not be removed. Here we report the development of in silico models of glycosylated His\(_6\)-FLAG-hIFN\(\gamma \) fusion proteins and employ long-scale molecular dynamics (MD) simulations to explain these unexpected experimental results and to study in detail the effect of the tags and glycosylation on the structure and dynamics of the fusion glycoproteins.