title: Intermediate activity of midge antifreeze protein is due to a tyrosine-rich ice-binding site and atypical ice plane affinity.
abstract: An antifreeze protein (AFP) from a midge (Chironomidae) was recently discovered and modelled as a tightly wound disulfide-braced solenoid with a surface-exposed rank of stacked tyrosines. New isoforms of the midge AFP have been identified from RT-PCR and are fully consistent with the model. Although they differ in the number of 10-residue coils, the row of tyrosines that form the putative ice-binding site is conserved. Recombinant midge AFP has been produced, and the properly folded form purified by ice affinity. This monomeric AFP has a distinct circular dichroism spectrum, a melting temperature between 35 and 50 °C and is fully renaturable on cooling. Mutagenesis of the middle tyrosine in the rank of seven eliminates antifreeze activity, whereas mutation of a tyrosine off this predicted ice-binding face had no such effect. This AFP has unusual properties compared to other known AFPs. First, its freezing-point depression activity is intermediate between that of the hyperactive and moderately active AFPs. As with hyperactive AFPs, when midge AFP-bound ice crystals exceed their freezing-point depression, ice grows explosively perpendicular to the c-axis. However, midge AFP does not bind to the basal plane of ice as do hyperactive AFPs, but rather to a pyramidal plane that is at a shallower angle relative to the basal plane than binding planes of moderate AFPs. These properties distinguish midge AFP from all other ice-binding proteins and the intermediate activity level fits well to the modest challenge of protecting newly emerged adult insects from late spring frosts.
  
  
  METHODS
  Nucleotide sequences of new midge AFP isoforms are available in the GenBank database under accession numbers KU094814-8. Sequences will be released after publication.
authors.affiliation: Protein Function Discovery Group, Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada.
authors.forename: Koli
authors.initials: K
authors.lastname: Basu
authors.affiliation: Department of Biochemistry, Programme in Cell Biology, Hospital for Sick Children, University of Toronto, Canada.
authors.forename: Samantha S
authors.initials: SS
authors.lastname: Wasserman
authors.affiliation: Protein Function Discovery Group, Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Jeronimo
authors.affiliation: Protein Function Discovery Group, Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada.
authors.forename: Laurie A
authors.initials: LA
authors.lastname: Graham
authors.affiliation: Protein Function Discovery Group, Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D021301:Antifreeze Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003460:Crystallization; D007053:Ice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D021301:Antifreeze Proteins; D007053:Ice; D020033:Protein Isoforms; D014443:Tyrosine
keywords: beta-solenoid fold; disulfide bonding; fluorescence-based ice plane affinity; freezing-point depression; ice-affinity purification