Antifreeze-Mechanistic Study through VT-EPR Spectral Analysis and ICE Growth Inhibition of Spin Labeled ICE Binding Proteins

Abstract

Species that inhabit extreme cold climates, survive by producing Ice Binding Proteins (IBPs). Through protein-binding to the surface of ice crystal nucleus, inhibition to the ice crystal growth is achieved which prevents cell damage. This study focused on type 1 IBPs from the winter flounder. The mechanism of ice growth inhibition was studied by variable temperature dependent electron paramagnetic resonance (VT-EPR) techniques on spin-labeled type I IBPs at different sites along its α-helical structure. The spin-labeled IBPs were formed through labeling the cysteine substituted IBPs. The EPR spectral lineshapes were analyzed by multicomponent EPR lineshape simulation to obtain the correlation times of the spin labels in the surrounding environments. Cryo-microscopy was used to observe the ice crystals. The changes in antifreeze activities and shapes of the ice crystals by the spins labeled IBPs were compared with the wild type IBP. We will discuss the results including observations of the spin labeled IBP binding to ice seed crystals, EPR spectra, and simulations. Categorization of molecular motion and correlation times surrounding the spin labeled groups were observed in anisotropic, slow, intermediate, and fast tumbling conditions, which revealed the water/ice surroundings along the different sites of the spin labeled IBPs at various temperatures.