title: 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.
authors.affiliation: Departments of Biochemistry and Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Marshall
authors.forename: Melanie M
authors.initials: MM
authors.lastname: Tomczak
authors.forename: Sherry Y
authors.initials: SY
authors.lastname: Gauthier
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kuiper
authors.forename: Christopher
authors.initials: C
authors.lastname: Lankin
authors.forename: Virginia K
authors.initials: VK
authors.lastname: Walker
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021341:Antifreeze Proteins, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005399:Fishes; D005615:Freezing; D009036:Moths; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry; D010473:Perciformes; D011485:Protein Binding / Q000235:genetics; D012996:Solutions; D013712:Tenebrio; D013997:Time Factors; D000509:alpha-Fetoproteins / Q000737:chemistry
chemical_list: D021301:Antifreeze Proteins; D021341:Antifreeze Proteins, Type II; D009211:Myoglobin; D012996:Solutions; D000509:alpha-Fetoproteins; C089666:antifreeze protein, sea raven