Structural basis for the appearance of a molten globule state in chimeric molecules derived from lysozyme and alpha-lactalbumin.

The problem as to why alpha-lactalbumin, in the absence of Ca(2+), forms a molten globule intermediate, in contrast to its structural homologue lysozyme, has been addressed by the construction of chimeras of human lysozyme in which either the Ca(2+)-binding loop or a part of helix C of bovine alpha-lactalbumin were transplanted. Previously, we have shown that the introduction of both structural elements together in the lysozyme matrix causes the apo form of the resulting chimera to display molten globule behavior during the course of thermal denaturation. In this article, we demonstrate that this molten globule character is not correlated with the Ca(2+)-binding loop. Also, the Del 101 mutant in which Arg101 was deleted to simulate the alpha-lactalbumin conformation of the connecting loop between helix C and helix D, does not show a stable equilibrium intermediate. Rather, the molten globule character of the chimeras has to be related with a specific part of helix C. More particularly, attention is drawn to the four hydrophobic side-chains I93, V96, I99, and L100, the lysozyme counterparts of which are constituted of less bulky valines and alanine. Our observations are discussed in terms of decreased stability of the native form and increased stability of the intermediate molten globule.

Kinetic and equilibrium intermediate states are different in LYLA1, a chimera of lysozyme and alpha-lactalbumin.

For several proteins, a striking resemblance has been observed between the equilibrium partially folded state and the kinetic burst-phase intermediate, observed just after the dead-time in refolding experiments. This has led to the general statement that the conformation of both types of intermediates is similar. We show, at least for one of the proteins investigated here, that, although both states have some common characteristics, they are not identical. LYLA1 is a chimeric protein resulting from the transplantation of the Ca(2+)-binding loop and the adjacent helix C of bovine alpha-lactalbumin into the homologous position (residues 76-102) in human lysozyme. The apo-form of LYLA1 unfolds through a partially folded state, in analogy with the folding behaviour of the structurally homologous alpha-lactalbumin. The folding kinetics of LYLA1 and of its wild-type homologue, human lysozyme, are investigated by means of stopped-flow fluorescence and CD spectroscopy. In the case of human lysozyme, refolding involves parallel pathways as indicated by experiments in the presence of a fluorescent inhibitor. For apo-LYLA1, the burst-phase intermediate is compared with the equilibrium intermediate. At neutral pH, both states correspond, in that an important amount of secondary structure has been established, but the burst-phase intermediate is shown to be significantly less stable than the equilibrium intermediate. At pH 1.85, in the presence of 1.5 M guanidinium hydrochloride (GdnHCl) and at 25 degrees C, the equilibrium partially folded state of LYLA1 is 100% populated. When LYLA1 is rapidly diluted from 6 M GdnHCl to 1.5 M under these conditions, a time-dependent evolution of the fluorescence signal is observed, reflecting the transition from a burst-phase to a different equilibrium intermediate. These results provide strong evidence for the non-identity of both states in this protein.

A simplified purification procedure of alpha-lactalbumin from milk using Ca(2+)-dependent adsorption in hydrophobic expanded bed chromatography.

The technique of expanded bed adsorption is originally designed for a direct recovery of proteins from fermentor feedstocks. In this article we describe the use of expanded bed adsorption for the recovery of alpha-lactalbumins from defatted milk using the hydrophobic Streamline Phenyl gel. alpha-Lactalbumins are Ca(2+)-binding proteins. Upon Ca2+ removal, they undergo a significant conformation change rendering them more hydrophobic. Based on this unique property we develop a protocol for fast and efficient purification of alpha-lactalbumin from milk. The use of this technique results in a reduction of the number of chromatographic purification steps.

Equilibrium and kinetic folding of pigeon lysozyme.

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.

Interaction of detergents with bovine lens alpha-crystallin: evidence for an oligomeric structure based on amphiphilic interactions.

We have studied the quaternary structure of alpha-crystallin in the presence of increasing concentrations of amphiphilic and neutral detergents using gel filtration, light-scattering, boundary and equilibrium sedimentation. We observed a continuous reduction of the molar mass of the polymeric alpha-crystallin on increasing the concentration of sodium dodecyl sulphate from 0.1 mM to 5 mM, ending up with the monomeric peptides. Dodecyltrimethylammonium bromide also disrupts the oligomeric structure of alpha-crystallin but the interaction appears to be cooperative: in the sharp transition region (for a 1 mg/ml protein solution) from 3 to 8 mM of the detergent, only the native protein and a mixture of monomeric and dimeric peptide-DTAB complexes can be observed. Concomitant studies of the circular dichroism in the far UV revealed a substantial decrease of the beta-sheet and increase of the alpha-helix secondary structure. The latter can be related to the presence of amphiphilic polypeptide sequences in the constituent alpha A and alpha B peptides. These studies reveal for the first time a direct relation between changes in the secondary structure of the alpha A and alpha B peptides and the formation of the oligomeric alpha-crystallin structure: the binding of the amphiphilic detergent reduces the beta-sheet content, induces the formation of alpha-helix secondary structure and reduces the tendency of the peptide to form large aggregates. The different mechanisms for reducing the oligomeric size by anionic and cationic detergents with identical apolar parts stresses the importance of charge interactions. Our findings support some aspects of the micelle model of alpha-crystallin and can be related to its chaperone activity.

Conformational stability of LYLA1, a synthetic chimera of human lysozyme and bovine alpha-lactalbumin.

LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca(2+)-binding site and helix C) of bovine alpha-lactalbumin has been inserted. The equilibrium unfolding of this hybrid protein has been examined by circular dichroism and tryptophan fluorescence techniques. The reversible denaturation process induced by temperature or by addition of chemical denaturant is three-state in the case of apo-LYLA1 and two-state in the presence of Ca2+. The Ca(2+)-bound form of the chimera exhibits higher stability than both wild-type lysozyme and alpha-lactalbumin. The stability of the apo-form, however, is intermediate between that of the parent molecules. Unfolding of apo-LYLA1 involves an intermediate state that becomes populated to a different extent under various experimental conditions. Combination of circular dichroism with bis-ANS fluorescence experiments has permitted us to characterize the acid state of LYLA1 as a molten globule. Furthermore our results strongly suggest the presence of multiple denatured states depending on external conditions.

A Ca(2+)-binding chimera of human lysozyme and bovine alpha-lactalbumin that can form a molten globule.

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.

An equilibrium partially folded state of human lysozyme at low pH.

Temperature-induced unfolding of human lysozyme has been monitored by circular dichroism and by nuclear magnetic resonance experiments at a variety of low pH values. The results indicate that, although at pH values above 3 unfolding appears to be consistent with a two-state model, at lower pH values this is not the case. At pH 1.2, for example, unfolding of the tertiary structure occurs at a temperature approximately 10 deg. C lower than that of the secondary structure. At 60 degrees C there is no detectable native tertiary structure remaining for human lysozyme at pH 1.2, although far-UV CD results show preservation of some 40% of the signal attributable to alpha-helical elements in the protein. This indicates the existence of a partially folded state of human lysozyme at low pH that has at least some characteristics of the well-defined molten globule state of the homologous alpha-lactalbumins and of the kinetic intermediates observed in the folding of alpha-lactalbumins and of c-type lysozymes. These results suggest that the absolute distinction between these two groups of proteins in terms of their different unfolding behaviour is not valid, and provide insights into possible features stabilizing such states.

Partially folded states of equine lysozyme. Structural characterization and significance for protein folding.

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)

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.

Hydrophobic interaction of lysozyme and alpha-lactalbumin from equine milk whey.

From fluorescence measurements on mixtures of bis-ANS and equine lysozyme and from Ca(2+)-dependent hydrophobic interaction chromatography of equine lysozyme, it is demonstrated that Ca2+ binding induces a conformational change upon which hydrophobic regions in the protein become less accessible. Bis-ANS fluorescence titrations in the absence of Ca2+ and in 2 mM Ca2+ are also performed with equine alpha-lactalbumin variants B and C. These variants differ by an amino-acid exchange Asp----Ile at residue 95. The fluorescence titration curves indicate that the accessibility of the probe to the Ca2+ conformers is clearly influenced by the mutation. The Ca(2+)-dependent exclusion of a hydrophobic domain is used in a new and simplified method for preparing lysozyme and alpha-lactalbumins simultaneously from equine milk whey.

Conformational aspects of the Cu2+ binding to alpha-lactalbumin. Characterization and stability of the Cu-bound state.

By circular dichroism experiments the existence of a typical Cu2(+)-bound state is demonstrated for bovine- and for goat alpha-lactalbumin. As in the near-UV region an important ligand to metal charge-transfer band overlaps with the aromatic band of the protein, a subtraction method is developed in order to determine the net effect of Cu2+ ions on the protein conformation. The Cu2(+)-bound state, characterized by a vanishing tertiary structure and a substantial loss of secondary structure, clearly differs from the well-known Ca2(+)-, apo-, and acid conformers. At room temperature, the Cu2+ binding has already decreased the alpha-helix content of bovine alpha-lactalbumin to the extent that further unfolding by thermal or guanidine hydrochloride denaturation behaves in a non-cooperative way. Since for goat alpha-lactalbumin the Cu2+ binding to His-68 is much less important than for bovine alpha-lactalbumin, we observe a somewhat different conformational behaviour for goat alpha-lactalbumin. The results of this conformational circular dichroism study are confirmed by isothermal calorimetric data.

Stability of equine lysozyme. I. Thermal unfolding behaviour.

The thermal denaturation of Ca(2+)- and apo-forms of equine lysozyme was followed by using far and near UV circular dichroism and intrinsic fluorescence methods. The difference found between the temperature dependence of the ellipticity at 222 nm and 287 nm, which show two stages in the thermal transition, and those at 228 nm and 294 nm, which indicate only one stage over a wide range of temperatures reflects that different subdivisions of the protein molecule are characterized by a different stability, cooperativity and pathway of denaturation. The first transition, reflected in the increase of the ellipticity at 222 nm and 287 nm, coincides with the transition detected by fluorescence and occurs at 30-50 degrees C for the apo-form and at 50-60 degrees C for the Ca(2+)-form of lysozyme. It seems to correlate with the transfer of some tryptophan residues to a more hydrophobic environment and with a local rearrangement of the tertiary and secondary structures. The unfolding transition detected by the decrease of the ellipticity at all wavelengths occurs nearly in the same temperature region for the apo- and Ca(2+)-forms, i.e. 50-80 degrees C and 55-80 degrees C, respectively. The presence of a Ca(2+)-binding loop in equine lysozyme may be partly responsible for the drastic destabilization of its structure as a whole both in the presence but especially in the absence of Ca2+ in comparison with hen and human lysozymes.

Thermodynamic data on the binding of six M2(+)-ions to bovine, goat, and human alpha-lactalbumin.

By batch microcalorimetry we titrated the apo-forms of bovine, goat, and human alpha-lactalbumin with Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, and Cd2+ ions at pH 7.5 and 25 degrees C. The titration curves enabled us to calculate the apparent enthalpy changes and binding constants and thus, also the free energy and the entropy changes of the binding. CD-spectra showed that all cations induce the same conformational change to the native form of the protein. The calorimetric and spectroscopic results, as well as sequence comparisons confirm the hypothesis that all these ions occupy the very same site on the molecule. The thermodynamic parameters, plotted vs the ionic radii, run parallel for the three proteins, which illustrates the earlier proposed "rigid site" model.