Tetra-n-butylammonium bromide forms the title semi-clathrate hydrate crystal, C16H36N+.Br-.38H2O, under atmospheric pressure. The cation and anion lie at sites with mm symmetry and seven water molecules lie at sites with m symmetry in space group Pmma. Br- anions construct a cage structure with the water molecules. Tetra-n-butylammonium cations are disordered and are located at the centre of four cages, viz. two tetrakaidecahedra and two pentakaidecahedra in ideal cage structures, while all the dodecahedral cages are empty.

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis . Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

The psychrophilic fungi Coprinus psychromorbidus and Typhula ishikariensis produced unique antifreeze proteins (AFPs) in the extracellular space. Molecular masses of purified fungal AFPs of C. psychromorbidus and T. ishikariensis were approximately 22 and 23 kDa, respectively. Cloned genes of AFPs from T. ishikariensis do not have any similarity with known proteins. Purified fungal AFPs from cultural filtrate of T. ishikariensis and recombinant fungal AFP from methylotrophic yeast formed specific ice crystals resembling "Stone Age knives". These observations indicate that fungal AFPs do not form proper hexagonal ice crystals to inhibit their growth and that fungal AFPs can probably bind to surfaces of ice crystals in an irregular manner.Key words: antifreeze protein, snow mold fungi, Coprinus psychromorbidus, Typhula ishikariensis.

We used directed evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. By combining random point mutagenesis, saturation mutagenesis, and DNA shuffling, a thermostable variant, Xyl^st, was identified which contained three amino acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature (the midpoint of the melting curves) for the Xyl^st variant compared with the wild-type enzyme after incubation for 10 min was elevated from 58 to 68 °C. At 60 °C the wild-type enzyme was inactivated within 5 min, but Xyl^st retained full activity for at least 2 h. The stabilization was accompanied by evidence of thermophilicity; that is, an increase in the optimal reaction temperature from 55 to 65 °C and lower activity at low temperatures and higher activity at higher temperatures relative to wild type. To elucidate the mechanism of thermal stabilization, three-dimensional structures were determined for the wild-type and Xyl^st enzymes. A cavity was identified around Gln-7/Asn-8 in wild type that was filled with bulky, hydrophobic residues in Xyl^st. This site was not identified by previous approaches, but directed evolution identified the region as a weak point. Formation of an intermolecular disulfide bridge via Cys-179 was observed between monomers in Xyl^st. However, the stability was essentially the same in the presence and absence of a reducing agent, indicating that the increased hydrophobicity around the Cys-179 accounted for the stability.

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.