Stability effects associated with the introduction of a partial and a complete Ca(2+)-binding site into human lysozyme.
Two mutants of human lysozyme were synthesized. Mutant A92D, in which Ala92 was substituted by Asp, contains a partial Ca(2+)-binding site and mutant M4, in which Ala83, Gln86, Asn88 and Ala92 were replaced by Lys, Asp, Asp and Asp respectively, contains the complete Ca(2+)-binding site of bovine alpha-lactalbumin. The Ca(2+)-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 Ca(2+)-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.
Haezebrouck
P
P
Interdisciplinary Research Center, Katholieke Universiteit Leuven, Kortrijk, Belgium.
De Baetselier
A
A
Joniau
M
M
Van Dael
H
H
Rosenberg
S
S
Hanssens
I
I
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D005822:Genetic Vectors; D006358:Hot Temperature; D006801:Humans; D007768:Lactalbumin / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D007768:Lactalbumin; D009113:Muramidase; D002118:Calcium