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Article

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

Supporting Info

Brad L. Pentelute^†, Zachary P. Gates^†, Jennifer L. Dashnau^‡, Jane M. Vanderkooi^‡ and Stephen B. H. Kent^†^§

Department of Chemistry, Department of Biochemistry & Molecular Biology, and Institute for Biophysical Dynamics, Gordon Center for Integrative Research, University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, and Department of Biochemistry and Biophysics,909 Stellar Chance Building, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059

J. Am. Chem. Soc., 2008, 130 (30), pp 9702–9707

DOI: 10.1021/ja801352j

Publication Date (Web): July 4, 2008

†

Department of Chemistry and Institute for Biophysical Dynamics, Gordon Center for Integrative Research, University of Chicago.

‡

University of Pennsylvania.

Department of Biochemistry and Molecular Biology, Gordon Center for Integrative Research, University of Chicago.

Abstract

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.

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