STRUCTURES OF HEAT-STABLE AND UNSTABLE HOMOLOGS 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 Levl. and determined its amino acid sequenc e [Liu, X., Maeda, S., Hu, Z., Aiuchi, T., Nakaya, K. & Kurihara, Y. ( 1993) Eur.: J. Biochem. 211, 281-287] and the disulfide structure [Nir asawa, S., Liu, X., Nishino, T. and Kurihara, Y. (1993) Biochim, Bioph ys. Acm 1202, 277-280]. We have now purified four additional homologue s of mabinlin. The sweet activities of mabinlin III and mabinlin TV we re unchanged by incubation for Ih at 80 degrees C, as was found previo usly for mabinlin II, while the sweet activity of mabinlin I-1 was com pletely abolished by a 1-h incubation at 80 degrees C. The circular di chroic spectrum showed that ex-helical structures of mabinlins II-IV w ere unchanged by the 1-h incubation at 80 degrees C, while the ex-heli cal structures of mabinlin I-1 were completely destroyed by the I-h in cubation in parallel with the decrease of the sweet activity. To compa re the structures of the heat-stable and unstable homologues, we deter mined their amino acid sequences and the disulfide array. The position s of four disulfide bridges of mabinlin I-1 were the same as those of mabinlin II, suggesting that the disulfide bridges do not contribute t o 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 a nd mabinlin III. A-chain residue at position 32 was lacking in mabinli n IV and the A-chain residue at position 22 was identical in both mabi nlin I-1 and mabinlin II. The B-chain residue at position 47 was the o nly 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 st position 47; the difference in the heat-stable homologues is due to the presence of an arginine re sidue and the difference of the unstable homologue is due to the prese nce 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 stabi lity.