Partitioning of Fish and Insect Antifreeze Proteins into Ice Suggests They Bind with Comparable Affinity

Abstract

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.

Modified Langmuir isotherm for a two-domain adsorbate: Derivation and application to antifreeze proteins

“…The activity increase is attributed to an increased affinity for the surface by increasing the binding face or size of the protein [43,44]. Point mutations have also been shown to affect the thermal hysteresis by reducing the geometric fit of the protein to the ice surface [45,46]. This also is attributable to a change in the affinity for the ice surface.…”

Hyperactive Antifreeze Protein from Winter Flounder Is a Very Long Rod-like Dimer of α-Helices

“…The fractions from this region were pooled (total volume of 200 ml), and the AFP(s) they contained were further purified by three successive rounds of ice affinity purification (IAP) using a temperature gradient of Ϫ1 to Ϫ5°C over 30 h (13,14). In this technique, a hemisphere of ice is slowly grown in a protein solution.…”

Molecular modalities of insect cold survival: current understanding and future trends

“…Fish have five different classes of AFPs, each with preferential binding to a specific surface of the growing ice crystal lattice [67]. Insect AFPs have not been subdivided into as many classes, but evidence shows insect AFPs bind to ice with comparable affinity and greater effectiveness than fish AFPs as measured by the degree of thermal hysteresis [68]. AFP activity is enhanced by protein enhancers [69], polyols, and organic anions [70].…”