H. Van Dael

The thermotropic transition of large unilamellar (LUV) vesicles of dimyristoyl phosphatidylcholine by Raman spectroscopy

Abstract By observing Raman spectra in the 2800–3000 cm −1 region, we determined the gel to liquid transition characteristics of large unilamellar vesicles of dimyristoyl phosphatidylcholine. This transition occurs between 22°C and 28.5°C. The large unilamellar vesicles can be well distinguished from the small unilamellar vesicles, which gel to liquid transition is spread out from 10°C to 27°C. The multilamellar vesicles on the other hand melt in a highly cooperative phenomenon at 23.7°C. The intermediate character of the transition of large unilamellar vesicles is also illustrated by data of the lateral order parameter.

Tyrosine group behaviour in bovine α-lactalbumin as revealed by its Raman effect

Side group behaviour is often used for conformational studies of proteins. We have performed Raman spectroscopic measurements on the tyrosine groups of bovine α-lactalbumin. The 850/830 cm-1 doublet intensity ratio is a direct measure of the negative charge state of the phenolic oxygen and of the tyrosine environment. pH measurements confirm the existence of an acid conformer of BLA, that is comparable to, but clearly distinguishable from the apo-conformer. Following the Siamwiza theory, the Tyr groups in this partially unfolded state are situated in a more hydrophobic environment. Observation of Tyr groups behaviour in the denaturated states obtained by thermal or chemical treatment leads us to the same conclusion. However, the behavior of tryptophan groups is quite different. In an unfolded sate, the Trp residues are mostly exposed to the solvent.The stabilizing role of Ca2+ and Na+ ions in BLA is also investigated.

The interaction of phenol with lipid bilayers

The depression of the phase-transition temperature of dimyristoyl- and dipalmitoylphosphatidylcholine vesicles induced by phenol has been investigated by fluorescence polarization. This effect is strongly pH and concentration dependent. Only the uncharged phenol molecule influences the fluidity of the bilayer so that the interaction of phenol with the bilayer can be situated in the hydrophobic acyl chain region. Direct measurements of the partitioning of phenol in the phospholipid vesicles confirm these results and show a limited and concentration-dependent solubility. Phase-transition temperature depressions, obtained from thermodynamic analysis of partition coefficient measurement, are in good agreement with the experimental values.

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway.Abstract. Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway.

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.

A circular dichroic study of Cu(II) binding to bovine α-lactalbumin

Abstract The visible and ultraviolet circular dichroic spectra resulting from the interaction of bovine α-lactalbumin with successive Cu(II) ions have been recorded under a variety of conditions. Analysis of the observed charge-transfer and d-d band transitions can be made in terms of two kinds of binding sites: at a histidyl group and at the N-terminal amino group, respectively. At basic pH the amide nitrogens of the peptide backbone progressively take part in the coordination. The occupation of the high affinity calcium binding site by Ca(II) and Mn(II) does not influence the Cu(II) binding process, suggesting that there is no direct interaction between this site and the Cu(II) binding sites.

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.

Thermodynamics of Mn2+-binding to goat α-lactalbumin

AbstractBy means of reaction calorimetry we measured the apparent enthalpy change, ΔHapp, of the binding of Mn2+-ions to goat α-lactalbumin as a function of temperature. The observed ΔHapp can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat α-lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (ΔH, ΔG, ΔS, ΔCp) into the binding and conformational contributions. By circular dichroism we showed that NH4+-ions, upon binding to bovine a-lactalbumin, induce the same conformational change as do Na+ and K+: the binding constant

An equilibrium and a kinetic stopped-flow fluorescence study of the binding of various metal ions to goat alpha-lactalbumin.

K_{NH_4^ + }^{app}