Adsorption rates measurements of an insect antifreeze protein to ice

Abstract

Antifreeze proteins (AFPs), a subset of ice-binding proteins (IBP), are found in various types of organisms that survive sub-freezing temperatures. AFPs adsorb to the surface of endogenous ice crystals and inhibit their growth, thereby preventing freezing injuries and death. The activity of an AFP is quantified by measuring the thermal hysteresis (TH) activity, which is the gap between the melting and freezing points created by the adsorption of the protein to the ice surface. GrAFP, an AFP found in a primitive arthropod, consists of nine polyproline type II (PPII) helix bundles. The helix bundle assembly allows for easy manipulation of the ice-binding site (IBS), which can impact the activity of the protein. GrAFP relies on hydrogen bonding to achieve its native fold, which leads to thermal instability. TmAFP, a protein stabilized by disulfide bonds rather than hydrogen bonding, has a higher TH activity compared to GrAFP and this can be due to the presence of stronger disulfide bonds. The adsorption rate of AFPs to ice (Kon) positively correlates with the TH activity of AFPs. In this thesis, Fluorescence microscopy coupled to coldstages and fluorescent-labelled AFPs were used to measure the adsorption rates of GrAFP to ice at various concentrations in an ultimate goal to obtain the value of Kon. Comparing the adsorption rate of GrAFP obtained here (0.010 μM-1s-1) with that of AFGP1-5 (0.013 μM-1s-1) shows that AFGP1-5 has a slightly higher rate. However, in the presence of borate, which deactivates the glycoprotein, GrAFP has a higher rate. The adsorption rate of AFPIII from an eelpout (0.008 μM- 1s-1) is also lower than that GrAFP. These results provide important insights into the structureactivity relationship of AFPs and illustrate the correlation between the TH activity of an AFP and its adsorption rate. Future experiments include measuring the adsorption rates for mutant GrAFP with different IBS sizes to better understand this structural effect on the TH activity, which increases with the size of the IBS.