Crystallographic studies of neuraminidase-sialic acid complexes indicate that sialic acid is distorted on binding the enzyme. Three arginine residues on the enzyme interact with the carboxylate group of the sugar which is observed to be equatorial to the saccharide ring as a consequence of its distorted geometry. The glycosidic oxygen is positioned within hydrogen-bonding distance of Asp-151, implicating this residue in catalysis.

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The amino acid sequence of a methionine-rich 2s seed protein from sunflower (Heliunthus annuus L.) and the sequence of a cDNA clone which codes for the entire primary translation product have been determined. The mature protein consists of a single polypeptide chain of 103 amino acids (molecular mass 12133 Da) which contains 16 residues of methionine and 8 residues of cysteine. The cDNA sequence established that the protein is synthesized as a precursor of 141 residues with a typical hydrophobic signal sequence of 25 residues followed by a further 13-residue hydrophobic pro-sequence which is presumably removed by post-translational cleavage. The sequence of the mature protein and that deduced from the cDNA were identical with no evidence of processing at the C-terminus. Comparison of the sunflower methionine-rich protein sequence with sequences of other seed 2s proteins from dicotyledons and monocotyledons showed limited but distinct sequence similarities; in particular the arrangement of the cysteine residues was conserved. The sunflower protein shows 34% identity with the methionine-rich Brazil nut 2s protein and the prepro regions of the precursors of these two proteins show about 50% identity. This similarity indicates that these methionine-rich 2s proteins have diverged as a subclass of the 2s superfamily of proteins which contain only 2-3% methionine. While the related 2s proteins from other dicotyledons are processed to a small and large subunit, the sunflower protein is not cleaved in this way.The seed storage proteins of most dicotyledonous plants contain two major protein classes, globulins and albumins, which are distinguished on the basis of solubility [l]. These proteins are expressed during seed development as precursors which undergo co-and post-translational modification prior to deposition in protein bodies [2]. The globulins, which are soluble in high-salt buffers and have sedimentation coefficients of 7s and 11S, are characterized by high levels of arginine, glutamine and asparagine, which provide a source of nitrogen for the developing seedling [2].The albumins are a more diverse group of proteins, usually soluble in water, with sedimentation coefficients of approximately 2s and some are rich in cysteine. While some 2s proteins appear to have only a storage function, some others such as lectins and protease and a-amylase inhibitors have biological activity. As a consequence of this these proteins are nutritionally undesirable. The sequences of a number of 2s seed storage proteins from different plants have shown that some of these proteins share sequence similarities and they have been grouped into a 2s superfamily of related sulfur- (EC 3.4.21.19).Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X56686. The novel amino acid sequence data have been deposited with the EMBL sequence data bank. rich seed proteins [3]. The 2s proteins from castor bean [4], rapeseed [5], mustard seed [6] Brazi...

The single-chain antibody variable fragment (scFv), with a 15-residue polypeptide linker (Gly,Ser),, of monoclonal antibody NClO was expressed in Escherichia coli and purified to homogeneity. This scFv molecule, refolded from 6 M guanidine hydrochloride, was predominantly a monomer of 27 kDa and was stable on storage at 4" and 20°C. At higher protein concentrations (= 5 mg/ml) dimer and higher-molecular-mass multimers were formed and freezing enhanced this aggregation. The dimer was not stable and dissociated to monomer at 20°C with a half-life of approximately 8 days. The higher-molecular-mass multimers and dimer dissociated to monomer in 60% ethylene glycol. Both the monomer and dimer were active and with tern N9 sialidase yielded complexes of 276 kDa and 569 kDa, respectively, indicating that four scFv molecules boundkialidase tetramer and that the dimer was bivalent and cross-linked two sialidase tetramers. Binding studies at low concentrations and using radiolabelled scFv indicated that the binding affinity of the dimer was approximately twofold higher than that of the monomer, and the binding affinities of the scFv were similar to that of the parent NClO antigen-binding fragment (Fab) molecule. A complex between tern N9 sialidase and NClO scFv was crystallized and the structure of the complex was solved at 0.3-nm resolution by X-ray diffraction. Comparison of this scFv/sialidase structure with the parent Fabhalidase structure revealed that the modes of attachment of scFv and Fab to sialidase were very similar. There was no discernible electron density for the peptide linker joining the variable heavy (V,) and variable light (V,) chains. A close interaction between two symmetryrelated scFv suggests that they may have crystallized as dimers.Monoclonal antibodies with their unique specificity and affinity are used widely as immunodiagnostic and therapeutic reagents [I] A scFv fragment of antibody NC10, which recognizes an epitope on influenza virus sialidase, was recently cloned and expressed in Escherichia coli [14]. The V, and V, domains were linked with the 15-residue peptide described above and also contained a hydrophilic octapeptide (FLAG) [19] at the C-terminus of the V, domain as an affinity label to aid in detection of the scFv. The purified, monomeric scFv was found to form dimers and higher-molecular-mass aggregates at a protein concentration greater than 5 mg/ml. Recently, the presence of dimers and higher-molecular-mass multimers