Structural investigation, computational analysis, and theoretical cryoprotectant approach of antifreeze protein type IV mutants

Antifreeze proteins (AFPs) have unique features to sustain life in sub-zero environments due to ice recrystallization inhibition (IRI) and thermal hysteresis (TH). AFPs are in demand as agents in cryopreservation, but some antifreeze proteins have low levels of activity. This research aims to improve the cryopreservation activity of an AFPIV. In this in silico study, the helical peptide afp1m from an Antarctic yeast AFP was modeled into a sculpin AFPIV, to replace each of its four α-helices in turn, using various computational tools. Additionally, a new linker between the first two helices of AFPIV was designed, based on a flounder AFPI, to boost the ice interaction activity of the mutants. Bioinformatics tools such as ExPASy Prot-Param, Pep-Wheel, SOPMA, GOR IV, Swiss-Model, Phyre2, MODFOLD, MolPropity, and ProQ were used to validate and analyze the structural and functional properties of the model proteins. Furthermore, to evaluate the AFP/ice interaction, molecular dynamics (MD) simulations were executed for 20, 100, and 500 ns at various temperatures using GROMACS software. The primary, secondary, and 3D modeling analysis showed the best model for a redesigned antifreeze protein (AFP1mb, with afp1m in place of the fourth AFPIV helix) with a QMEAN (Swiss-Model) Z score value of 0.36, a confidence of 99.5%, a coverage score of 22%, and a p value of 0.01. The results of the MD simulations illustrated that AFP1mb had more rigidity and better ice interactions as a potential cryoprotectant than the other models; it also displayed enhanced activity in limiting ice growth at different temperatures.