The Thr- and Ala-rich hyperactive antifreeze protein from inchworm folds as a flat silk-like β-helix

Biochemistry. 2011 May 31;50(21):4467-78. doi: 10.1021/bi2003108. Epub 2011 May 4.


Inchworm larvae of the pale beauty geometer moth, Campaea perlata, exhibit strong (6.4 °C) freezing point depression activity, indicating the presence of hyperactive antifreeze proteins (AFPs). We have purified two novel Thr- and Ala-rich AFPs from the larvae as small (∼3.5 kDa) and large (∼8.3 kDa) variants and have cloned the cDNA sequences encoding both. They have no homology to known sequences in current BLAST databases. However, these proteins and the newly characterized AFP from the Rhagium inquisitor beetle both contain stretches rich in alternating Thr and Ala residues. On the basis of these repeats, as well as the discontinuities between them, a detailed structural model is proposed for the 8.3 kDa variant. This 88-residue protein is organized into an extended parallel-stranded β-helix with seven strands connected by classic β-turns. The alternating β-strands form two β-sheets with a thin core composed of interdigitating Ala and Ser residues, similar to the thin hydrophobic core proposed for some silks. The putative ice-binding face of the protein has a 4 × 5 regular array of Thr residues and is remarkably flat. In this regard, it resembles the nonhomologous Thr-rich AFPs from other moths and some beetles, which contain two longer rows of Thr in contrast to the five shorter rows in the inchworm protein. Like that of some other hyperactive AFPs, the spacing between these ice-binding Thr residues is a close match to the spacing of oxygen atoms on several planes of ice.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry*
  • Amino Acid Sequence
  • Animals
  • Antifreeze Proteins / chemistry*
  • Antifreeze Proteins / genetics
  • Antifreeze Proteins / isolation & purification
  • Base Sequence
  • Chromatography, Affinity
  • Circular Dichroism
  • DNA Primers
  • DNA, Complementary
  • Expressed Sequence Tags
  • Larva / chemistry*
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Moths / growth & development*
  • Polymerase Chain Reaction
  • Protein Folding
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Threonine / chemistry*


  • Antifreeze Proteins
  • DNA Primers
  • DNA, Complementary
  • Threonine
  • Alanine