Mirror Image Forms of Snow Flea Antifreeze Protein Prepared by Total Chemical Synthesis Have Identical Antifreeze Activities

• Published in: Journal of the American Chemical Society Vol. 130; no. 30; pp. 9702 - 9707
• Main Authors: Pentelute, Brad L, Gates, Zachary P, Dashnau, Jennifer L, Vanderkooi, Jane M, Kent, Stephen B. H
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 30.07.2008; Amer Chemical Soc
• Subjects: Amino Acid Sequence; Animals; Antifreeze Proteins - chemical synthesis; Antifreeze Proteins - chemistry; Chemistry; Chemistry, Multidisciplinary; Circular Dichroism; Contraindications; Disulfides - chemistry; Ice; Molecular Sequence Data; Peptide Fragments - chemical synthesis; Peptide Fragments - chemistry; Physical Sciences; Protein Folding; Protein Structure, Tertiary; Science & Technology; Siphonaptera - chemistry; Stereoisomerism; PRESERVATION; LIGATION; RECRYSTALLIZATION; PHASE PEPTIDE-SYNTHESIS; ICE BINDING; POLYPEPTIDE
• ISSN: 0002-7863; 1272-7863; 1520-5126
• DOI: 10.1021/ja801352j

The recently discovered glycine-rich snow flea antifreeze protein (sfAFP) has no sequence homology with any known proteins. No experimental structure has been reported for this interesting protein molecule. Here we report the total chemical synthesis of the mirror image forms of sfAFP (i.e., l-sfAFP, the native protein, and d-sfAFP, the native protein’s enantiomer). The predicted 81 amino acid residue polypeptide chain of sfAFP contains Cys residues at positions 1, 13, 28, and 43 and was prepared from four synthetic peptide segments by sequential native chemical ligation. After purification, the full-length synthetic polypeptide was folded at 4 °C to form the sfAFP protein containing two disulfides. Chemically synthesized sfAFP had the expected antifreeze activity in an ice recrystallization inhibition assay. Mirror image d-sfAFP protein was prepared by the same synthetic strategy, using peptide segments made from d-amino acids, and had an identical but opposite-sign CD spectrum. As expected, d-sfAFP displays the same antifreeze properties as l-sfAFP, because ice presents an achiral surface for sfAFP binding. Facile synthetic access to sfAFP will enable determination of its molecular structure and systematic elucidation of the molecular basis of the antifreeze properties of this unique protein.