Structures of heat‐stable and unstable homologues of the sweet protein mabinlin.

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Abstract
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°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°C. The circular dichroic spectrum showed that α‐helical structures of mabinlins II–IV were unchanged by the 1‐h incubation at 80°C, while the α‐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 homologues. 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. It is possible that a salt bridge, formed between a at B‐chain arginine residue at position 47 and the caroxylic group of an A‐chain or B‐chain C‐terminal residue contributes to the heat stability.