Comparison of the Cu2+ binding to bovine, goat and human alpha-lactalbumin.

Cu2+ binds to bovine alpha-lactalbumin at two different sites, principally at a hystidyl residue and in second instance at a deprotonated amide group. In human alpha-lactalbumin, that is lacking His 68, only the second binding site was observed, so that evidence is given that His 68 in bovine alpha-lactalbumin is responsible for the major Cu2+ binding. In goat alpha-lactalbumin, the histidyl binding effectively occurs but only to a lesser degree as the accessibility of His 68 is reduced by the greater compactness of goat alpha-lactalbumin. In the three species the Cu2+ binding is independent on the occupation of the primary Ca(2+)- site.     

Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin

Comparative studies of the unfolding equilibria of two homologous proteins, bovine alpha-lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of alpha-lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein. In previous studies, alpha-lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of alpha-lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, alpha-lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the featureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+, the unfolding transition of alpha-lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native alpha-lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.     

The perturbations of the native state of goat alpha-lactalbumin induced by 1,1'-bis(4-anilino-5-naphthalenesulfonate) are Ca2+-dependent.

In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition. Therefore, it can be expected that in both conditions the protein is equally susceptible to interaction with bis-ANS. Nevertheless, we have observed a number of interesting differences in the interaction of the dye with the apo and Ca2+ form. Native apo-GLA binds two bis-ANS molecules and in the complex with bis-ANS, the far-UV circular dichroism (CD) spectrum of apo-GLA becomes similar to that of the protein in the molten globule state. In contrast, native Ca2+-GLA binds five bis-ANS molecules and the far-UV CD spectrum of native Ca2+-GLA is conserved for the complex. In both cases, the high activation energies observed in kinetic experiments indicate that upon binding, large parts of the protein structure have to be reorganized. The reduced perturbation of the protein structure in the presence of Ca2+ can be attributed to local stabilization effects.     

Effect of metal ion binding on the secondary structure of bovine alpha-lactalbumin as examined by infrared spectroscopy

We have examined the influence of monovalent and divalent cations on the secondary structure of bovine alpha-lactalbumin at neutral pH using Fourier-transform infrared spectroscopy. Our present studies are based on previously reported amide I' component band assignments for this protein [Prestrelski, S. J., Byler, D. M., & Thompson, M. P. (1991) Int. J. Pept. Protein Res. 37, 508-512]. The results indicate that upon dissolution, alpha-lactalbumin undergoes a small, but significant, time-dependent conformational change, regardless of the ions present. Additionally, these studies provide the first quantitative measure of the well-known secondary structural change which accompanies calcium binding. Results indicate that removal of Ca2+ from holo alpha-lactalbumin results in local unfolding of the Ca(2+)-binding loop; the spectra indicate that approximately 16% of the backbone chain changes from a rigid coordination complex to an unordered loop. We have also examined the effects of binding of several other metal ions. Our studies have revealed that binding of Mn2+ to apo alpha-lactalbumin (Ca(2+)-free), while inducing a small, but significant, conformational change, does not cause the alpha-lactalbumin backbone conformation to change to that of the holo (Ca(2+)-bound) form as characterized by infrared spectroscopy. Similar changes to those induced by Mn2+ are observed upon binding of Na+ to apo alpha-lactalbumin, and furthermore, even at very high concentrations (0.2 M), Na+ does not stabilize a structure similar to the holo form. Binding of Zn2+ to the apo form of alpha-lactalbumin does not result in significant backbone conformational changes, suggesting a rigid Zn(2+)-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein.     

Kinetics of folding and unfolding of goat alpha-lactalbumin

The equilibrium unfolding and the kinetic folding and unfolding of goat alpha-lactalbumin (GLA) were studied by near- and far-ultraviolet circular dichroism (CD) and by stopped-flow fluorescence spectroscopy. Specifically, the influence of environmental conditions such as pH and Ca2+ binding was examined. Compared to the apo-form, the Ca2+-bound form was found to be strongly stabilized in equilibrium conditions at pH 7.5 and 25 degrees C. The kinetics of the refolding of apo-GLA show a major change of fluorescence intensity during the experimental dead-time, but this unresolved effect is strongly diminished in holo-GLA. In both cases, however, the chevron plots can adequately be fitted to a three-state model. Moreover, double-mix stopped-flow experiments showed that the native state (N) is reached through one major pathway without the occurrence of alternative tracks. In contrast to the homologous bovine alpha-lactalbumin (BLA), the compactness of GLA is strongly influenced by the presence of Ca2+ ions. Unlike the two-state transition observed in guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation experiments at higher pH, an equilibrium intermediate state (I) is involved in denaturation at pH 4.5. In the latter case, analysis of the kinetic data makes clear that the intermediate and the unfolded states (U) show practically no Gibbs free energy difference and that they are in rapid equilibrium with each other. A possible explanation for these variations in stability and in folding characteristics with pH could be the degree of protonation of His107 that directly influences non-native interactions. Variation of environmental conditions and even small differences in sequence, therefore, can result in important effects on thermodynamic and folding parameters.     

Kinetic and structural characterization of adsorption-induced unfolding of bovine alpha -lactalbumin.

Conformational changes of bovine alpha-lactalbumin induced by adsorption on a hydrophobic interface are studied by fluorescence and circular dichroism spectroscopy. Adsorption of bovine alpha-lactalbumin on hydrophobic polystyrene nanospheres induces a non-native state of the protein, which is characterized by preserved secondary structure, lost tertiary structure, and release of calcium. This partially denatured state therefore resembles a molten globule state, which is an intermediate in the folding of bovine alpha-lactalbumin. Stopped-flow fluorescence spectroscopy reveals two kinetic phases during adsorption with rate constants k(1) approximately 50 s(-1) and k(2) approximately 8 s(-1). The rate of partial unfolding is remarkably fast and even faster than unfolding induced by the addition of 5.4 m guanidinium hydrochloride to native alpha-lactalbumin. The large unfolding rates exclude the possibility that unfolding of bovine alpha-lactalbumin to the intermediate state occurs before adsorption takes place. Stopped-flow fluorescence anisotropy experiments show that adsorption of bovine alpha-lactalbumin on polystyrene nanospheres occurs within the dead time (15 ms) of the experiment. This shows that the kinetic processes as determined by stopped-flow fluorescence spectroscopy are not affected by diffusion or association processes but are solely caused by unfolding of bovine alpha-lactalbumin induced by adsorption on the polystyrene surface. A scheme is presented that incorporates the results obtained and describes the adsorption of bovine alpha-lactalbumin.     

Conversion of human alpha-lactalbumin to an apo-like state in the complexes with basic poly-amino acids: toward understanding of the molecular mechanism of antitumor action of HAMLET

It was recently shown that alpha-lactalbumin associated with oleic acid (HAMLET) interacts with core histones thereby triggering apoptosis of tumor cells (J. Biol. Chem. 2003, 278, 42131). In previous work, we revealed that monomeric alpha-lactalbumin in the absence of fatty acids can also interact with histones and, moreover, with basic poly-amino acids (poly-Lys and poly-Arg) that represent simple models of histone proteins (Biochemistry 2004, 43, 5575). Association of alpha-lactalbumin with histone or poly-Lys(Arg) essentially changes its properties. In the present work, the character of the changes in structural properties and conformational stability of alpha-lactalbumin in the complex with poly-Lys(Arg) has been studied in detail by steady-state fluorescence, circular dichroism, and differential scanning calorimetry. Complex formation strongly depends on ionic strength, confirming its electrostatic nature. Experiments with the poly-amino acids of various molecular masses demonstrated a direct proportionality between the number of alpha-lactalbumin molecules bound per poly-Lys(Arg) and the surface area of the poly-amino acid random coil. The binding of the poly-amino acids to Ca2+-saturated human alpha-lactalbumin decreases its thermal stability down to the level of its free apo-form and decreases Ca2+-affinity by 4 orders of magnitude. The conformational state of alpha-lactalbumin in a complex with poly-Lys(Arg), named alpha-LActalbumin Modified by Poly-Amino acid (LAMPA), differs from all other alpha-lactalbumin states characterized to date, representing an apo-like (molten globule-like) state with substantially decreased affinity for calcium ion. The requirement for efficient conversion of alpha-lactalbumin to the LAMPA state is a poly-Lys(Arg) chain consisting of several tens of amino acid residues.     

Divalent cation-induced changes in conformation of protein kinase C

Using physical techniques, circular dichroism and intrinsic and extrinsic fluorescence, the binding of divalent cations to soluble protein kinase C and their effects on protein conformation were analyzed. The enzyme copurifies with a significant concentration of endogenous Ca2+ as measured by atomic absorption spectrophotometry, however, this Ca2+ was insufficient to support enzyme activity. Intrinsic tryptophan fluorescence quenching occurred upon addition to the soluble enzyme of the divalent cations, Zn2+, Mg2+, Ca2+ or Mn2+, which was irreversible and unaffected by monovalent cations (0.5 M NaCl). Far ultraviolet (200-250 nm) circular dichroism spectra provided estimations of secondary structure and demonstrated that the purified enzyme is rich in alpha-helices (42%) suggesting a rather rigid structure. At Ca2+ or Mg2+ concentrations similar to those used for fluorescence quenching, the enzyme undergoes a conformational transition (42-24% alpha-helix, 31-54% random structures) with no significant change in beta-sheet structures (22-26%). Maximal effects on 1 microM enzyme were obtained at 200 microM Ca2+ or 100 microM Mg2+, the divalent cation binding having a higher affinity for Mg2+ than for Ca2+. The Ca2(+)-induced transition was time-dependent, while Mg2+ effects were immediate. In addition, there was no observed energy transfer for protein kinase C with the fluorescent Ca2(+)-binding site probe, terbium(III). This study suggests that divalent cation-induced changes in soluble protein kinase C structure may be an important step in in vitro analyses that has not yet been detected by standard biochemical enzymatic assays.     

Effects of a helix substitution on the folding mechanism of bovine alpha-lactalbumin

The structure, stability, and unfolding-refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105-110) comes from equine lysozyme and the remainder of the sequence comes from bovine alpha-lactalbumin were studied by circular dichroism spectroscopy and stopped-flow measurements, and the results were compared with those of bovine alpha-lactalbumin. The substitution of the flexible loop in bovine alpha-lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine alpha-lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine alpha-lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non-native interaction in the flexible loop region and that the disruption of the non-native interaction reduces the free energy barrier of refolding. We conclude that the non-native interaction in the molten globule state may act as a kinetic trap for the folding of alpha-lactalbumin.     

The allosteric mechanism of bovine liver glutamate dehydrogenase. Evidence from circular-dichroism studies for a conformational change in the ternary complex enzyme-(oxidized nicotinamide-adenine dinucleotide)-glutarate.

1. Computer averaging of multiple scans was used to refine the circular dichroism spectrum of bovine liver glutamate dehydrogenase, revealing well-defined structure in the aromatic region. 2. The circular dichroism of NAD+ bound to glutamate dehydrogenase is strongly negative at 260nm, probably owing to immobilization of the adenosine moiety. Loss of the characteristic adenine-nicotinamide interaction suggests that the coenzyme is bound in an unstacked conformation. 3. Glutarate and succinate, substrate analogues that are both inhibitors competitive with glutamate, do not significantly perturb the circular-dichroism spectrum of the enzyme in the absence of NAD+. 4. In the presence of NAD+, 150nM-succinate decreases the negative circular dichroism corresponding to bound coenzyme, but does not affect the protein circular dichroism. However, ISOmM-glutarate causes profound alternations of the circular-dichroism spectra of the bound NAD+ and of the enzyme, indicative of a protein conformational change. This direct evidence of conformational change specifically promoted by C5 dicarboxylates confirms the previous inference from protection studies. 5. The conformational change is discussed in relation to the allosteric mechanism of glutamate dehydrogenase.     

Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations

Folding reaction of goat alpha-lactalbumin has been studied by stopped-flow circular dichroism and molecular dynamics simulations. The effects of four single mutations and a double mutation on the stability of the protein under a native condition were studied. The mutations were introduced into residues located at a hydrophobic core in the alpha-domain of the molecule. Here we show that an amino acid substitution (T29I) increases the native-state stability of goat alpha-lactalbumin against the guanidine hydrochloride-induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild-type and mutant goat alpha-lactalbumin measured by stopped-flow circular dichroism showed that the local structure around the Thr29 side chain was not constructed in the transition state of the folding reaction. To characterize the local structural change around the Thr29 side chain to an atomic level of resolution, we performed high-temperature (at 400 K and 600 K) molecular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 portion of the molecule experienced structural disruption accompanied with the loss of inter-residue contacts and with the water molecule penetration in the 400-K simulation as well as in four of the six 600-K simulations. Disruption of the N-terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous report.     

Structural changes of alpha-lactalbumin induced by low pH and oleic acid

The effects of low pH and oleic acid on conformation and association state of Ca2+-depleted bovine alpha-lactalbumin (apo-BLA) have been studied by electrospray ionization mass spectrometry, fluorescence spectroscopy, and circular dichroism. The experimental results demonstrate that two structurally distinct species exist in the conformational transition of apo-BLA induced by low pH. One species populates at pH 3.0 characterized as a monomeric molten globule state and the other accumulates at pH 4.0-4.5 which is a partially folded dimer. Oleic acid promotes the formation of the dimeric intermediate at pH 4.0 and 7.0, but increases the content of molten globule state remarkably at pH 3.0 compared with that in the absence of oleic acid, indicating that oleic acid at pH 3.0 plays a different role from those at pH 4.0 and 7.0. Our data provide insight into the mechanism of pH-dependent and oleic acid-dependent structural changes and oligomerization of alpha-lactalbumin, and will be helpful to the understanding of the apoptosis-inducing function of multimeric alpha-lactalbumin in which oleic acid is a necessary cofactor.     

Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.     

Molecular biology of copper. A circular dichroism study on copper complexes of thionein and penicillamine.

Chicken liver Cd, Zn-thionein (metallothionein) was isolated from Cd-pretreated chickens weighing 1 500 g. The native Cd, Zn-thionein contained 9 g-atoms of metals per 12 000 g of protein. Upon the addition of Cu(CH3CN)4ClO4, all Cd2 and Zn2 were successfully replaced. 15 g-atoms of Cu from the acetonitrile perchlorate complex were bound to the protein. Due to the absence of aromatic amino acid residues, thionein has unique ultraviolet and circular dichroism properties. The shoulder of the ultraviolet spectrum at 250 nm (A250 X A280(-1) = 23.9) was shifted to 275 nm (A250 X A280(-1) = 1.6). No significant absorption was detected in the visible region. Th conformational changes of the protein moiety were much more visible in the circular dichroism spectra. The titration with Cu(CH3CH)2 caused the appearence of three new Cotton effects: 257.5 nm (+), 350 nm (+) and 301 nm (-). The negative Cotton effect at 239 nm of the original metallothionein was completely levelled off. The binding strength of copper with thionein is extraordinarily high: it survives proton treatment up to pH 1.9. Displacement of the Cd2 by Cu employing Cd-thionein which was formed at pH 2.2 resulted in the same circular dichroism properties as observed for Cu-thionein. D-Penicillamine proved a suitable model for the metal-free thionein, since redox reactions and polymerization of the sterically hindered thiol residue are known to be slow. The correlation of the circular dichroism properties of either copper complex using thionein or D-penicillamine was surprisingly high. Circular dichroism measurements of Cu(I)-D-penicillamine revealed Cotton effects at 255 nm (+), 280 nm (+) and 355 nm (-). Upon examining the red-violet mixed Cu(-i)-cu(II)-D-penicillamine complex, Cotton bands in the visible region at 425 nm (-) and 495 nm (+) were seen. In many blue copper enzymes, the copper is assumed to be in the neighborhood of both cysteine and aromatic amino acid residues, which are known to play an important role in the electron transfer. This is not the case in the Cu-thionein, which would explain many different properties of this copper protein. It is very attractive to conclude that the sterically hindered SH-group of D-penicillamine reacts with excess copper in a specific way, similar to the Cu-thionein. This phenomenon could explain the considerable success of D-penicillamine in the treatment of Wilson's disease.     

The interaction of peripheral proteins and membranes studied with alpha-lactalbumin and phospholipid bilayers of various compositions

To characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine alpha-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMR-monitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent alpha-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of alpha-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane.     

Secondary structure of proteins from circular dichroism spectra. V. Secondary structure of proteins in a "molten globule" state

A method that accounts for the contribution made by aromatic amino acid residues in circular dichroism spectra of proteins has been used in order to analyze the structure of bovine carboanhydrase B, bovine and human alpha-lactalbumin in the native state and when denatured with acid and temperature. At acid- and temperature-induced transitions of the secondary structure of these proteins has been shown not to change. However the rigidity of their tertiary structure decreases (the environment of aromatic amino acid residues is made more symmetrical).     

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.     

Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin

To discuss the relation between the folding mechanism and the chemical structure of proteins, the reversible unfolding reactions of human alpha-lactalbumin by acidification and by guanidine hydrochloride at 25 degrees C are studied by means of circular dichroism, difference spectra and pH-jump measurements and are compared with those for bovine alpha-lactalbumin. As shown previously for bovine alpha-lactalbumin, the folding process at neutral pH is not explained by a simple two-state mechanism but involves an intermediate form that has the same amount of helical structures as the native form. The transition between the intermediate and the fully denatured states is too rapid to be measured and corresponds to the helix-coil transition of the backbone. One of the differences of human alpha-lactalbumin from the bovine protein is the remarkable stability of the intermediate at neutral pH, which can be explained by differences in the primary chemical structure. Another difference is the existence at acid pH of an additional helical form, which is more helical than the native form. The transition from this to the intermediate or to the fully denatured one also is shown to resemble the helix-coil transition. The following folding scheme of human alpha-lactalbumin is proposed: formula: (see text). Here N is the native form, and the intermediate is a macroscopic state distributed around the state A3 at neutral pH, while the distribution in the acid and fully denautured states shifts toward Am and A-n, respectively.     

Purification, calcium-binding properties, and conformational studies on a 28-kDa cholecalcin-like protein from bovine brain

A large-scale preparation method for bovine brain 28-kDa cholecalcin-like protein is described. Flow dialysis binding studies revealed that the protein binds at least 3 mol of Ca2+/mol of protein. The protein undergoes conformational changes on binding calcium as shown by UV differential absorption spectroscopy, near and far UV circular dichroism, and intrinsic fluorescence. Circular dichroism (CD) studies in the far UV indicate an apparent increase in helical content in the presence of Ca2+. The effect of calcium on the protein structure is nearly maximum for 1 Ca2+ bound/protein molecule. UV differential absorption studies on the binding of the Ca2+ agonist Tb3+ and Tb3+ luminescence induced by energy Trp----Tb3+ transfer indicate that Tb3+ binds to two higher affinity Ca2+-binding sites. These sites are probably very close to the single Trp residue. Analysis of the fluorescence parameters of the single tryptophan residue in the apoprotein and its accessibility to ionic and neutral quenchers suggests that this residue is located in a highly hydrophobic domain on the protein surface.