X-ray Structure of Snow Flea Antifreeze Protein Determined by Racemic Crystallization of Synthetic Protein Enantiomers

• Published in: Journal of the American Chemical Society Vol. 130; no. 30; pp. 9695 - 9701
• Main Authors: Pentelute, Brad L, Gates, Zachary P, Tereshko, Valentina, Dashnau, Jennifer L, Vanderkooi, Jane M, Kossiakoff, Anthony A, Kent, Stephen B. H
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 30.07.2008
• Subjects: 60 APPLIED LIFE SCIENCES; Amino Acid Sequence; Animals; ANTIFREEZE; Antifreeze Proteins - chemistry; BASIC BIOLOGICAL SCIENCES; CRYSTALLIZATION; CRYSTALLOGRAPHY; Crystallography, X-Ray; INSECTS; MIXTURES; Molecular Sequence Data; Protein Conformation; PROTEIN STRUCTURE; PROTEINS; RESIDUES; Siphonaptera - chemistry; Stereoisomerism; STRUCTURAL MODELS; SYNTHESIS; WAVELENGTHS
• ISSN: 0002-7863; 1272-7863; 1520-5126
• DOI: 10.1021/ja8013538

Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 Å. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.