High-resolution three-dimensional structures are now available for four of seven non-homologous sh and insect antifreeze proteins (AFPs). For each of these structures, the ice-binding site of the AFP has been de ned by site-directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice-binding proteins shows that they have the following attributes in common. The binding sites are relatively at and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic-more so than that portion of the protein exposed to the solvent. Surface-surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts.

Type I antifreeze protein (AFP) from winter flounder is an alanine-rich, 37 amino acid, single K K-helix that contains three 11 amino acid repeats (Thr-X 2 -Asx-X 7 ), where X is generally Ala. The regularly spaced Thr, Asx and Leu residues lie on one face of the helix and have traditionally been thought to form hydrogen bonds and van der Waals interactions with the ice surface. Recently, substitution experiments have called into question the importance of Leu and Asn for ice-binding. Sequence alignments of five type I AFP isoforms show that Leu and Asn are not well conserved, whereas Ala residues adjacent to the Thr, at right angles to the Leu/Asn-rich face, are completely conserved. To investigate the role of these Ala residues, a series of Ala to Leu steric mutations was made at various points around the helix. All the substituted peptides were fully K K-helical and remained as monomers in solution. Wild-type activity was retained in A19L and A20L. A17L, where the substitution lies adjacent to the Thr-rich face, had no detectable antifreeze activity. The nearby A21L substitution had 10% wildtype activity and demonstrated weak interactions with the ice surface. We propose a new ice-binding face for type I AFP that encompasses the conserved Ala-rich surface and adjacent Thr.z 1999 Federation of European Biochemical Societies.

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Some cold water marine fishes avoid cellular damage because of freezing by expressing antifreeze proteins (AFPs) that bind to ice and inhibit its growth; one such protein is the globular type III AFP from eel pout. Despite several studies, the mechanism of ice binding remains unclear because of the difficulty in modeling the AFP-ice interaction. To further explore the mechanism, we have determined the x-ray crystallographic structure of 10 type III AFP mutants and combined that information with 7 previously determined structures to mainly analyze specific AFP-ice interactions such as hydrogen bonds. Quantitative assessment of binding was performed using a neural network with properties of the structure as input and predicted antifreeze activity as output. Using the cross-validation method, a correlation coefficient of 0.60 was obtained between measured and predicted activity, indicating successful learning and good predictive power. A large loss in the predictive power of the neural network occurred after properties related to the hydrophobic surface were left out, suggesting that van der Waal's interactions make a significant contribution to ice binding. By combining the analysis of the neural network with antifreeze activity and x-ray crystallographic structures of the mutants, we extend the existing ice-binding model to a two-step process: 1) probing of the surface for the correct ice-binding plane by hydrogen-bonding side chains and 2) attractive van der Waal's interactions between the other residues of the ice-binding surface and the ice, which increases the strength of the protein-ice interaction. Many poikilothermic organisms have developed antifreeze proteins (AFPs)1 to resist freezing. Five classes of structurally diverse antifreeze proteins have been found in fish (for a review, see Ref. 1). These proteins act by adsorbing to the surface of ice and increasing the curvature of ice fronts between the bound AFPs (2). The freezing point at the surface is depressed, which in turn inhibits the growth of ice crystals. The difference between the temperature at which the ice begins to grow (burst point) and the temperature at which the ice crystal melts is known as thermal hysteresis (TH) and is used as a measure of AFP activity.Recently, several structures of type III AFP have been determined (4 -6). Based on the high-resolution x-ray structure (4), a model was proposed whereby surface adsorption occurs through a hydrogen bond match between the side chains of Gln-9, Asn-14, Thr-15, Thr-18, Gln-44, and the ice prism plane {1010}. These polar residues form part of a flat, amphipathic face that is thought to be the ice-binding surface (Fig. 1). However, the significance of the contribution from hydrogen bonds to the AFP-ice interaction has been questioned by several studies since then. A supposedly conservative change of Thr to Ser in type I AFP led to a large loss of TH activity, whereas a change to the hydrophobic residue valine, which is a better space-filling match, caused only a small loss (7,8). In the stud...

The large molecular size of antibody drugs is considered one major factor preventing them from becoming more efficient therapeutics. Variable regions of heavy chain antibodies (HCAbs), or single-domain antibodies (sdAbs), are ideal building blocks for smaller antibodies due to their molecular size and enhanced stability. In the search for better antibody formats for in vivo imaging and/or therapy of cancer, three types of sdAb-based molecules directed against epidermal growth factor receptor (EGFR) were constructed, characterized and tested. Eleven sdAbs were isolated from a phage display library constructed from the sdAb repertoire of a llama immunized with a variant of EGFR. A pentameric sdAb, or pentabody, V2C-EG2 was constructed by fusing one of the sdAbs, EG2, to a pentamerization protein domain. A chimeric HCAb (cHCAb), EG2-hFc, was constructed by fusing EG2 to the fragment crystallizable (Fc) of human IgG1. Whereas EG2 and V2C-EG2 localized mainly in the kidneys after i.v. injection, EG2-hFc exhibited excellent tumor accumulation, and this was largely attributed to its long serum half life, which is comparable to that of IgGs. The moderate size ($80 kDa) and intact human Fc make HCAbs a unique antibody format which may outperform whole IgGs as imaging and therapeutic reagents.Crown