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Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural determination of a gap junction channel composed of connexin 26 (Cx26) at 3.5 Å resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the aminoterminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal. [ABSTRACT FROM AUTHOR]

This article focuses on a study related to purification, complete amino acid sequence and structural characterization of the heat-stable sweet protein, mabinlin II. All experiments, except for the defatted proeedure, were carried out at 4°C. 400 mg decorticated seeds, free from pulps of the fruit, were ground into powder. The supernatant was colorless. Ammonium sulfate fractionation was carried out by addition of solid ammonium sulfate to the pooled solution to bring about 70% saturation. The eluted protein was monitored by absorption at 280 nm.

A new sweet protein, named mabinlin II, was extracted with 0.5 M NaCI solution from the seeds of Capparis masaikai Lévl. and purified by ammonium sulfate fractionation, carboxymethylcellulose- Sepharose ion-exchange chromatography and gel filtration. The sweetness of mabinlin II was unchanged by at least 48 h incubation at nearly boiling temperature. Purified mabinlin II thus obtained gave a single band having a molecular mass of 14 kDa on SDS/PAGE. In the presence of dithiothreitol, mabinlin II gave two bands having molecular masses of 4.6 kDa and 5.2 kDa on SDS/PAGE. Two peptides (A chain and B chain) were separated from reduced and S-carboxamidomethylated mabinlin II by HPLC. The amino acid sequences of the A chain and B chain were determined by the automatic Edman-degradation method. The A chain and B chain consist of 33-amino-acid and 72-amino-acid residues, respectively. The A chain is mostly composed of hydrophilic amino acid residues and the B chain also contains many hydrophilic residues. High similarity was found between the amino acid sequences of mabinlin 11 and 2S seed storage proteins, especially 2S albumin AT2S3 in Arabidopsis thaliana (mouse-ear cress). [ABSTRACT FROM AUTHOR]