Petra Haezebrouck

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.

A simplified purification procedure of α-lactalbumin from milk using Ca2+-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 α-lactalbumins from defatted milk using the hydrophobic Streamline Phenyl gel. α-Lactalbumins are Ca2+- 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 α-lactalbumin from milk. The use of this technique results in a reduction of the number of chromatographic purification steps.

Hydrophobic interaction of lysozyme and α-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 Ca 2+ binding induces a conformational change upon which hydrophobic regions in the protein become less accessible. Bis-ANS fluorescence titrations in the absence of Ca 2+ and in 2 mM Ca 2+ are also performed with equine α-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 Ca 2+ 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 α-lactalbumins simultaneously from equine milk whey.

INTERACTION OF DETERGENTS WITH BOVINE LENS ALPHA -CRYSTALLIN : EVIDENCE FOR AN OLIGOMERIC STRUCTURE BASED ON AMPHIPHILIC INTERACTIONS

Abstract We have studied the quaternary structure of α-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 α-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 α-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 β-sheet and increase of the α-helix secondary structure. The latter can be related to the presence of amphiphilic polypeptide sequences in the constituent αA and αB peptides. These studies reveal for the first time a direct relation between changes in the secondary structure of the αA and αB peptides and the formation of the oligomeric α-crystallin structure: the binding of the amphiphilic detergent reduces the β-sheet content, induces the formation of α-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 α-crystallin and can be related to its chaperone activity.

Equilibrium and kinetic studies on folding of canine milk lysozyme

Thermal and chemical unfolding studies of the calcium‐binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped‐flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo‐canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s−1. Furthermore, the results show that, unlike its drastic effect on the stability, Ca2+‐binding only marginally affects the refolding kinetics. During the refolding process of apo‐CL non‐native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non‐native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.

CONFORMATIONAL STABILITY OF LYLA1, A SYNTHETIC CHIMERA OF HUMAN LYSOZYME AND BOVINE ALPHA -LACTALBUMIN

Abstract LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca2+-binding site and helix C) of bovine α-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 Ca2+-bound form of the chimera exhibits higher stability than both wild-type lysozyme and α-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.

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

The problem as to why α‐lactalbumin, in the absence of Ca2+, 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 Ca2+‐binding loop or a part of helix C of bovine α‐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 Ca2+‐binding loop. Also, the Del 101 mutant in which Arg101 was deleted to simulate the α‐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. Proteins 2001;44:1–11.

Conformational aspects of the Cu2+ binding to α-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.

Unfolding Behaviour of Chimeric Proteins from Lysozyme and α-Lactalbumin

Lysozymes and α-lactalbumins are homologous proteins and are very similar with regard to sequence and structure. Nevertheless their unfolding behaviour and their conformational dynamics are fundamentally different. Whereas lysozymes unfold in a classical two-state process (N → U), α-lactalbumins unfold via an intermediate state, characterized as the molten globule state (N → MG → U). In addition, all α-lactalbumins bind Ca2+ tightly whereas this property is totally absent in lysozymes excepted in these lysozymes which show the same ligand residues as those found in α-lactalbumins.

Stability of Ca2+-binding mutants of human lysozyme

Abstract Yeast-expressed human lysozyme is found to show the same thermal denaturation behaviour as native human lysozyme. The A92D mutant has a somewhat lower transition temperature as the wild-type. Whilst not affecting the tertiary structure of this mutant, Ca2+ binding provokes a drastic change in its thermal characteristics when measured by circular dichroism in the far-UV region. The M4 mutant in the apo-form is slightly more stable than the wild-type and this stabilization effect is strongly enhanced by Ca2+ binding. Lys 83 seems to play an important role in this thermostability behaviour.