Stability Effects Associated With the Introduction of a Partial and a Complete Ca2+-binding Site Into Human Lysozyme
(1993) Protein Engineering — Vol. 6, n° 6, p. 643-649 (1993)
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- Two mutants of human lysozyme were synthesized. Mutant A92D, in which Ala92 was substituted by Asp, contains a partial Ca2+-binding site and mutant M4, in which Ala83, Gln86, Asn88 and Ala92 were replaced by Lys, Asp, Asp and Asp respectively, contains the complete Ca2+-binding site of bovine alpha-lactalbumin. The Ca2+-binding constants of wild type human lysozyme and of mutants A92D and M4, measured at 25-degrees-C and pH 7.5, were 2(+/- 1) x 10(2) M-1, 8(+/- 2) x 10(3) M-1 and 9(+/- 0.5) X 10(6) M-1 respectively. Information gathered from microcalorimetric and CD spectroscopic measurements indicates that the conformational changes of the M4 mutant lysozyme, induced by Ca2+ binding, are smaller than those observed for bovine alpha-lactalbumin and for the Ca2+-binding equine lysozyme. At pH 4.5, the thermostability of both the apo and Ca2+ forms of the A92D human was decreased in comparison with that of native human lysozyme. In particular, within the apo form of this mutant an alpha-helix-containing sequence was destabilized. In contrast, at the same pH the thermostability of the apo and Ca2+ forms of the M4 mutant lysozyme was increased. The epsilon-ammonium group of the Lys83 side chain is assumed to be responsible for the stabilization of the apo form of this mutant.
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Haezebrouck, P., Debaetselier, A., Joniau, M., Vandael, H., Rosenberg, S., & Hanssens, I. (1993). Stability Effects Associated With the Introduction of a Partial and a Complete Ca2+-binding Site Into Human Lysozyme. Protein Engineering, 6(6), 643-649. https://doi.org/10.1093/protein/6.6.643 (Original work published 1993)