Despite their homologous structure, c-type lysozymes and alpha-lactalbumins have been found to differ profoundly in their unfolding behavior, in that the alpha-lactalbumins readily enter a partially unfolded collapsed state (the "molten globule"), whereas lysozymes unfold cooperatively to a highly unfolded state. The calcium-binding property of lysozyme from equine milk provides an evolutionary link between the two families of proteins. We demonstrate here that equine lysozyme undergoes a two-stage unfolding transition upon heating or in the presence of guanidine hydrochloride that is highly dependent on the state of calcium binding. Differential scanning calorimetry shows the two transitions to be particularly well resolved in the calcium-free protein, where the first transition occurs with a midpoint at 44 degrees C at pH 4.5 or in 0.8 M GdnHCl at pH 7.5, 25 degrees C, and the second occurs near 70 degrees C at pH 4.5 or in 3.7 M GdnHCl at pH 7.5, 25 degrees C. In the presence of calcium, the first transition takes place with a midpoint of 55 degrees C or in excess of 2.5 M GdnHCl, but the parameters for the second transition remain unchanged. Fluorescence emission and UV difference absorption spectroscopy suggest that the first transition generates an intermediate state in which sequestration of some aromatic side chains from solvent has occurred whereas the second represents denaturation to a highly unfolded state. CD and 1H NMR results indicate that the intermediate state possesses extensive secondary and tertiary structure, although the latter is substantially disordered.(ABSTRACT TRUNCATED AT 250 WORDS)

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In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca(2+)-free form of the homologous alpha-lactalbumins forms an intermediate "molten globule" state. To understand this difference, we have produced a chimera of human lysozyme and bovine alpha-lactalbumin. In the synthetic gene of the former the sequence coding for amino acid residues 76-102 was replaced by that for bovine alpha-lactalbumin 72-97, which represents the Ca(2+)-binding loop and the central helix C. The chimeric protein, LYLA1, expressed in Saccharomyces cerevisiae was homogeneous on electrophoresis and mass spectrometry. Its Ca2+ binding constant was 2.50 (+/- 0.04) x 10(8) M-1, and its muramidase activity 10% of that of human lysozyme. One-dimensional NMR spectroscopy indicated the presence of a compact, well structured protein. From two-dimensional NMR spectra, main chain resonances for 118 of a total of 129 residues could be readily assigned. Nuclear Overhauser effect analysis and hydrogen-deuterium exchange measurements indicated the presence and persistence of all expected secondary structure elements. Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2+ form at 90 degrees C, whereas unfolding of the apo form occurred at 73 degrees C in the near-UV and 81 degrees C in the far-UV range. These observations illustrate that by transplanting the central part of bovine alpha-lactalbumin, we have introduced into human lysozyme two important properties of alpha-lactalbumins, i.e. stabilization through Ca2+ binding and molten globule behavior.

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In the present study, the search for a possible intermediate state in pigeon lysozyme is addressed by equilibrium and kinetic experiments using static and stopped-flow fluorescence and circular dichroism spectroscopies. In equilibrium conditions at pH 7.5, pigeon lysozyme shows no populated intermediate state in temperature- and GdnHCl-induced unfolding experiments. In the unfolding process at low pH, however, a distinct intermediate state with molten globule characteristics is observed. Ca2+ binding to the protein is found to stabilize the native state. The early folding intermediate observed in kinetic experiments corresponds to the equilibrium intermediate in that an important amount of secondary structure has already been established. Full accomplishment of native tertiary contacts is achieved in a fast exponential process with a rate constant (0.23-135 s-1) that is strongly dependent on refolding conditions. Binding experiments with the fluorescent inhibitor MeU-diNAG support these conclusions. The folding rate is not influenced by Ca2+ binding. Analysis of the refolding and unfolding kinetics determined as a function of denaturant concentration leads to a Gibbs energy profile with a rate-determining transition state between the N- and I-states. Comparison with previous results on the folding of hen egg white lysozyme emphasizes the crucial role of Trp 62 in stabilizing non-native interactions. The replacement of this residue by Tyr in pigeon lysozyme contributes to the formation of native tertiary contacts.

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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.

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