Utilizing Avidity To Improve Antifreeze Protein Activity: A Type III Antifreeze Protein Trimer Exhibits Increased Thermal Hysteresis Activity

Abstract

Antifreeze proteins (AFPs) are ice growth inhibitors that allow the survival of several species living at temperatures colder than the freezing point of their bodily fluids. AFP activity is commonly defined in terms of thermal hysteresis, which is the difference observed for the solution freezing and melting temperatures. Increasing the thermal hysteresis activity of these proteins, particularly at low concentrations, is of great interest because of their wide range of potential applications. In this study, we have designed and expressed one-, two-, and three-domain antifreeze proteins to improve thermal hysteresis activity through increased binding avidity. The three-domain type III AFP yielded significantly greater activity than the one- and two-domain proteins, reaching a thermal hysteresis of >1.6 °C at a concentration of <1 mM. To elucidate the basis of this increase, the data were fit to a multidomain protein adsorption model based on the classical Langmuir isotherm. Fits of the data to the modified isotherms yield values for the equilibrium binding constants for the adsorption of AFP to ice and indicate that protein surface coverage is proportional to thermal hysteresis activity.

Functional Analysis of a Bacterial Antifreeze Protein Indicates a Cooperative Effect between Its Two Ice-Binding Domains

“…As the number of accessible ice binding sites, or avidity, increases on an AFP, its efficiency at binding with ice and TH activity increase 32,33,44 . Hence, the cooperative effect we demonstrated between the two domains of IBPv may be a result of increasing avidity on each protein molecule.…”

From ice-binding proteins to bio-inspired antifreeze materials

“… 66 , 78 Maximal TH activities measured by cryoscopy range from 0.1–0.5 °C in apoplastic extracts from overwintering plants 4 , 61 and ∼2 °C for 30–40 g L –1 protein in fish serum 21 , 74 to ∼2 to 13 °C for 10-times lower concentrations in insect hemolymph. 54 TH activity can be improved upon addition of (sub)molar concentrations of small molecular solutes 79 and ions, 20 , 80 conjugation 81 to or complexation 82 with potentiating proteins 82 or other ice-binding proteins 81 and polymers, 83 , 84 and IBS enlargement. 85 However, salts like sodium borate can also inactivate AFGPs.…”

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

“…In both cases, connecting two almost homologous domains increases the TH value cooperatively compared with their monomeric AFPs [139,209]. Studies from the laboratories of Tsuda, Davies, and Holland have demonstrated that the multimerization of native type III AFP can increase TH activity [210,211,212]. Recently, Steven et al claimed the dendrimer-like AFPs showed higher TH values [213], and Phippen et al demonstrated 12 AFP-fused protein cage nanoparticles that increased the TH value to more than 50-fold that of monomeric AFP [214].…”