Structures of heat-stable and unstable homologues of the sweet protein mabinlin. The difference in the heat stability is due to replacement of a single amino acid residue.
There are several analogues of the sweet protein mabinlin. In previous studies, we purified the heat-stable analogue, mabinlin II, from the seeds of Capparis masaikai Lévl. and determined its amino acid sequence [Liu, X., Maeda, S., Hu, Z., Aiuchi, T., Nakaya, K. & Kurihara, Y. (1993) Eur. J. Biochem. 211, 281-287] and the disulfide structure [Nirasawa, S., Liu, X., Nishino, T. & Kurihara, Y. (1993) Biochim. Biophys. Acta 1202, 277-280]. We have now purified four additional homologues of mabinlin. The sweet activities of mabinlin III and mabinlin IV were unchanged by incubation for 1 h at 80 degrees C, as was found previously for mabinlin II, while the sweet activity of mabinlin I-1 was completely abolished by a 1-h incubation at 80 degrees C. The circular dichroic spectrum showed that alpha-helical structures of mabinlins II-IV were unchanged by the 1-h incubation at 80 degrees C, while the alpha-helical structures of mabinlin I-1 were completely destroyed by the 1-h incubation in parallel with the decrease of the sweet activity. To compare the structures of the heat-stable and unstable homologues, we determined their amino acid sequences and the disulfide array. The positions of four disulfide bridges of mabinlin I-1 were the same as those of mabinlin II, suggesting that the disulfide bridges do not contribute to the difference in the heat stability among the homologues. There was a high similarity among amino acid sequences of the homologoues. Only three amino acid residues (A-chain residues at positions 22 and 32 and B-chain residue at position 47) were different between mabinlin I-1 and mabinlin III. A-chain residue at position 32 was lacking in mabinlin IV and the A-chain residue at position 22 was identical in both mabinlin I-1 and mabinlin II. The B-chain residue at position 47 was the only residue present in all three heat-stable homologues (mabinlins II-IV) and is not present in the unstable homologue (mabinlin I-1). This suggests that the difference in the heat stability of mabinlin is due to the difference in a B-chain residue at position 47; the difference in the heat-stable homologues is due to the presence of an arginine residue and the difference of the unstable homologue is due to the presence of glutamine.(ABSTRACT TRUNCATED AT 400 WORDS)
Nirasawa
S
S
Department of Chemistry, Faculty of Education, Yokohama National University, Japan.
Nishino
T
T
Katahira
M
M
Uesugi
S
S
Hu
Z
Z
Kurihara
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004220:Disulfides / Q000737:chemistry; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012639:Seeds / Q000737:chemistry*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013549:Sweetening Agents / Q000737:chemistry*
D000596:Amino Acids; D004220:Disulfides; D010940:Plant Proteins; D013549:Sweetening Agents; C083584:mabinlin protein, Capparis masaikai