Peptide models of local and long-range interactions in the molten globule state of human alpha-lactalbumin

alpha-Lactalbumin, a small calcium-binding protein, forms an equilibrium molten globule state under a variety of conditions. A set of four peptides designed to probe the role of local interactions and the role of potential long-range interactions in stabilizing the molten globule of alpha-lactalbumin has been prepared. The first peptide consists of residues 20 through 36 of human alpha-lactalbumin and includes the entire B-helix. This peptide is unstructured in solution as judged by CD. The second peptide is derived from residues 101 through 120 and contains both the D and 310 helices. When this peptide is crosslinked via the native 28 to 111 disulfide to the B-helix peptide, a dramatic increase in helicity is observed. The crosslinked peptide is monomeric, as judged by analytical ultracentrifugation. The peptide binds 1-anilinonaphthalene-8-sulphonate (ANS) and the fluorescence emission maximum of the construct is consistent with partial solvent exposure of the tryptophan residues. The peptide corresponding to residues 101 to 120 adopts significant non-random structure in aqueous solution at low pH. Two hydrophobic clusters, one involving residues 101 through 104 and the other residues 115 through 119 have been identified and characterized by NMR. The hydrophobic cluster formed by residues 101 through 104 is still present in a smaller peptide containing only residues 101 to 111 of alpha-lactalbumin. The cluster also persists in 6 M urea. A non-native, pH-dependent interaction between the Y103 and H107 side-chains that was previously identified in the acid-denatured molten globule state was examined. This interaction was found to be more prevalent at low pH and may therefore be an example of a local interaction that stabilizes preferentially the acid-induced molten globule state.     

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.     

The chaperone-like alpha-crystallin forms a complex only with the aggregation-prone molten globule state of alpha-lactalbumin

The chaperone-like alpha-crystallin prevents aggregation of several proteins by interacting with their non-native states. Alpha-Lactalbumin adopts different non-native states under different experimental conditions. We have investigated the interaction of alpha-crystallin with three non-identical non-native states, using fluorescence, circular dichroism, and gel filtration chromatography. The compact molten globule state of apo-alpha-lactalbumin in tris buffer does not interact with alpha-crystallin. The expanded, flexible molten globule-like state of reduced apo-alpha-lactalbumin (formed at pH 7.2) also does not interact with alpha-crystallin. Only the aggregation-prone non-native state of reduced apo-alpha-lactalbumin formed at pH 6.0 interacts with alpha-crystallin to form a stable complex. The alpha-crystallin bound reduced apo-alpha-lactalbumin exhibits properties similar to those of a molten globule. Our results show that alpha-crystallin interacts only with the aggregation prone molten globule state of reduced apo-alpha-lactalbumin but not with the other non-aggregating molten globule states of the protein.     

The native-like tertiary fold in molten globule alpha-lactalbumin appears to be controlled by a continuous phase transition

On account of its ability to discriminate between secondary, loop and sidegroup structure and its special sensitivity to conformational mobility, vibrational Raman optical activity (ROA) has provided new insights into the complexity of order within the molten globule state from measurements on alpha-lactalbumin at pH 2.0 over the temperature range 2 to 45 degrees C. Thus while much of the secondary structure present in the native protein persists with only a small gradual decrease with increasing temperature, the tertiary backbone fold changes dramatically, being almost complete and native-like at 2 degrees C and almost completely disordered at 35 degrees C. The change of the tertiary fold with temperature is cooperative but has no latent heat, and so has the approximate characteristics of a continuous phase transition, being of the order-disorder type since it involves the interconversion of rigid, locally-ordered loop structure with disordered mobile backbone structure. This has implications for protein folding because the long-range correlations that exist in the critical region of a continuous (but not in a first-order) phase transition could resolve, in principle, the problem of how the protein finds its native-like folding pattern at the molten globule stage.     

Sequential unfolding of papain in molten globule state

Papain exhibits the characteristics of molten globule under acidic conditions as seen by circular dichroism, fluorescence and ANS binding. Between pH 2.0-2.5 the protein exhibits substantial secondary structure as indicated by far-UV CD spectrum but loses the persistent tertiary interactions of the native state. Enhanced binding of ANS to the state at pH 2.0 in relation to the native and unfolded states at neutral pH indicates a considerable exposure of aromatic side chains. Temperature and guanidine hydrochloride induced unfolding of papain in this state is noncooperative and the transition curves are biphasic in nature. As papain molecule consists of two domains, the results suggest that the domains unfold independently and sequentially.     

The herpes simplex virus triplex protein, VP23, exists as a molten globule

Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacent hexons and pentons in the herpes simplex virus type 1 capsid. VP23 was expressed in Escherichia coli and purified to homogeneity by Ni-agarose affinity chromatography. In vitro capsid assembly experiments demonstrated that the purified protein was functionally active. Its physical status was examined by differential scanning calorimetry, ultracentrifugation, size exclusion chromatography, circular dichroism, fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate binding studies. These studies established that the bacterially expressed VP23 exhibits properties consistent with its being in a partially folded, molten globule state. We propose that the molten globule represents a functionally relevant intermediate which is necessary to allow VP23 to undergo interaction with VP19C in the process of capsid assembly.     

Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule

The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.     

Determination of the volume changes for pressure-induced transitions of apomyoglobin between the native, molten globule, and unfolded states

The volume change for the transition from the native state of horse heart apomyoglobin to a pressure-induced intermediate with fluorescence properties similar to those of the well-established molten globule or I form was measured to be -70 ml/mol. Complete unfolding of the protein by pressure at pH 4.2 revealed an upper limit for the unfolding of the intermediate of -61 ml/mol. At 0.3 M guanidine hydrochloride, the entire transition from native to molten globule to unfolded state was observed in the available pressure range below 2.5 kbar. The volume change for the N-->I transition is relatively large and does not correlate well with the changes in relative hydration for these transitions derived from measurements of the changes in heat capacity, consistent with the previously observed lack of correlation between the m-value for denaturant-induced transitions and the measured volume change of unfolding for cooperativity mutants of staphylococcal nuclease (Frye et al. 1996. Biochemistry. 35:10234-10239). Our results support the hypothesis that the volume change associated with the hydration of protein surface upon unfolding may involve both positive and negative underlying contributions that effectively cancel, and that the measured volume changes for protein structural transitions arise from another source, perhaps the elimination of void volume due to packing defects in the structured chains.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

Conformational transitions provoked by organic solvents in beta-lactoglobulin: can a molten globule like intermediate be induced by the decrease in dielectric constant?

The macroscopic arrangement of the termination of the thoracic duct (TD) was examined in detail in Japanese cadavers, and the distribution of various types of collagen, tenascin, laminin, and fibronectin in the framework of the wall of the thoracic duct termination was demonstrated. We identified several branching patterns and pathways of the TD (i.e., those terminating 1) at the venous angle (Type A); 2) at the end of the internal jugular vein (Type B); 3) at the the external jugular vein (Type C); or 4) in a complex with various branches (Type D). The TD often divided into two or three trunks before or after passing beneath the left brachiocephalic vein. Compared with the main trunk of the TD, fewer muscle fibers, elastic fibers, specific compounds of the extracellular matrices, and valvular connective tissues were found near the termination of the TD and the valves of the TD and veins. Smooth muscle cells were arranged irregularly in each region of the TD.     

Structural characterization of the disulfide folding intermediates of bovine alpha-lactalbumin

Specific three- and two-disulfide intermediates that accumulate transiently during reduction of the disulfide bonds of Ca(2+)-bound bovine alpha-lactalbumin have been trapped, isolated, and characterized. The three-disulfide intermediate was shown to lack the Cys6-120 disulfide bond, confirming the observations of others. The newly-recognized two-disulfide form has been shown to lack the Cys6-120 and Cys28-111 native disulfide bonds. The remaining native disulfide bonds in the two partially reduced derivatives of alpha-lactalbumin are stable only when the proteins are in a Ca(2+)-bound state. Otherwise, they adopt an equilibrium between molten globule and unfolded conformations, and rapid thiol-disulfide interchange occurs, at a rate as high as when the proteins are fully unfolded in 8 M urea, to generate distinct mixtures of rearranged products. Urea gradient electrophoresis, circular dichroism, fluorescence, and ANS binding have been combined to give a detailed structural picture of alpha-lactalbumin, its derivatives with native and with nonnative disulfide bonds, and the fully reduced protein. The native structure of alpha-lactalbumin appears to be split by selective disulfide bond cleavage into at least one subdomain, which retains the Ca(2+)-binding site. The alpha-lactalbumin molten globule state is shown largely to result from nonspecific hydrophobic collapse, to be devoid of cooperative or specific tertiary interactions, and not to be stabilized substantially by the native or rearranged disulfide bonds.     

Production of bovine alpha-lactalbumin in the milk of transgenic pigs

High production of milk and its components are necessary to allow maximal growth of developing pigs. In this study, transgenic pigs were produced containing the alpha-lactalbumin gene, whose product is a potential limiting component in the production of milk. Two lines of transgenic pigs were produced to analyze the effects that overproduction of the milk protein alpha-lactalbumin may have on milk production and piglet growth. Transgenic pigs were produced through microinjection of the bovine alpha-lactalbumin gene. The gene construct contained 2.0 kb of 5' flanking region, the 2.0 kb coding region, and 329 bp of 3' flanking region. Sows hemizygous for the transgene produced as much as .9 g of bovine alpha-lactalbumin per liter of pig milk. The production of the bovine protein caused approximately a 50% increase in the total alpha-lactalbumin concentration of pig milk throughout a lactation. The concentration of bovine alpha-lactalbumin was highest on d 0 and 5 of lactation and decreased as lactation progressed. The ratio of bovine to porcine alpha-lactalbumin changed during the sow's lactation. This ratio was 4.3 to 1 on d 0 of lactation, but by d 20 of lactation the ratio was .43 to 1. This suggested that the bovine transgene and the endogenous porcine gene are under slightly different control mechanisms. The higher level of total alpha-lactalbumin present on d 0 of lactation was correlated with higher lactose percentage on d 0 in transgenic sows (3.8%), compared with controls (2.6%) (P < .01). Although there was also a trend for higher lactose percentage in transgenic sows on d 5 and 10 of lactation, no significant differences were observed. These data suggest that alpha-lactalbumin is limiting early in lactation of swine. Furthermore, higher concentrations of alpha-lactalbumin early in lactation may boost milk output.     

Characterization of a trifluoroethanol-induced partially folded state of alpha-lactalbumin

The protein alpha-lactalbumin exists in a partially folded molten globule state at pH 2.0, the A state. This state is believed to be compact, possessing a similar amount of secondary structure to the native state but having a flexible tertiary structure comprised mainly of non-specific hydrophobic clustering of residues. Addition of trifluoroethanol (TFE) to bovine, human and guinea pig alpha-lactalbumin at pH 2.0 has been found in each case to induce a conformational transition in the A state as monitored by circular dichroism, nuclear magnetic resonance chemical shifts, and 1-anilinonaphthalene-8-sulphonate binding. The mid-point of this transition is near 15% (v/v) TFE and is effectively complete by 50% (v/v) TFE at 315 K. Far ultraviolet circular dichroism ellipticities at 208 nm and 220 nm, usually taken as a measure of the degree of helical character, are substantially more negative in the TFE state than in the A state. Furthermore, backbone amide protons protected from solvent exchange in the A state are generally at least as strongly protected in the TFE state; patterns of protection appear similar in the two states and include at least part of both the B and C alpha-helices. One major difference from the A state is nevertheless evident: the ability to bind the fluorescent probe 1-anilinonaphthalene-8-sulphonate, characteristic of molten globule states, is lost in the TFE state. Like the A state, the TFE state of alpha-lactalbumin shows little chemical shift dispersion of side-chain resonances. Extensive line broadening in the nuclear magnetic resonance spectra, characteristic of slow conformational averaging in the A state, is, however, much reduced in the TFE state. The line narrowing observed in the TFE state has made it possible to obtain directly sequence-specific assignments for about 25% of the 123 residues of bovine alpha-lactalbumin in 50% (v/v) TFE. Two helices are amongst regions of structure so far identified from short-range backbone nuclear Overhauser enhancement (NOE) connectivities in two-dimensional spectra of the TFE state. One of the helices (residues 86 to 96) corresponds to the C-helix in the native structure. The other (residues 35 to 41) corresponds, however, to a region of the sequence that is not helical in the native state. The partially folded state of alpha-lactalbumin formed in TFE, therefore, supports both native and non-native secondary structure in the absence of persistent long-range tertiary structure.     

Kinetics of the homogeneous exchange of alpha-lactalbumin adsorbed on titanium oxide surface

BACKGROUND: We prospectively evaluated the potential of the 6-minute walk test compared with peak VO2 in predicting outcome of patients with New York Heart Association (NYHA) class II or III heart failure. METHODS AND RESULTS: Patients with a history of heart failure caused by systolic dysfunction were included. The combined final outcome (death or hospitalization for heart failure) was used as the judgment criterion. One hundred twenty-one patients (age 59+/-11 years; left ventricular ejection fraction 29.6%+/-13%) were included and followed for 1.53+/-0.98 years. Patients were separated into two groups according to outcome: group 1 (G1, 74 patients), without events, and group 2 (G2, 47 patients), who reached the combined end point. Peak VO2 was clearly different between G1 and G2 (18.5+/-4 vs. 13.9+/-4 ml/kg/min, p=0.0001) but not the distance walked (448+/-92 vs 410+/-126 m; p=0.084, not significant). Survival analysis showed that unlike peak VO2, the distance covered was barely distinguishable between the groups (p < 0.08). However, receiver operating characteristic curves revealed that the best performances for the 6-minute walk test were obtained for subjects walking < or =300 m. These patients had a worse prognosis than those walking farther (p=0.013). In this subset of patients, there was a significant correlation between distance covered and peak VO2 (r=0.65, p=0.011). Thus it appears that the more severely affected patients have a daily activity level relatively close to their maximal exercise capacity. Nevertheless, the 300 m threshold suggested by this study needs to be validated in an independent population. CONCLUSIONS: A distance walked in 6 minutes < or =300 m can predict outcome. Moreover, in these cases there is a significant correlation between the 6-minute walk test and peak VO2 demonstrating the potential of this simple procedure as a first-line screening test for this subset of patients.     

Functional identification of calcium binding residues in bovine alpha-lactalbumin

The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.     

Allergy to the heat-labile proteins alpha-lactalbumin and beta-lactoglobulin in mare's milk

BACKGROUND: Allergy to mare's milk is rare. Recently, however, mare's milk has been recommended for treatment of various ailments by practitioners of "alternative medicine," and it is available in health food stores. OBJECTIVE: We report a case of allergic reaction to mare's milk in a 51-year-old woman who was able to tolerate cow's milk. METHODS: The protein composition of mare's milk was determined by methods based on measurement of nitrogen content. The patient underwent prick and intracutaneous tests with commercially available bovine milk proteins and several mare's milk preparations, including mare's milk granulate and boiled mare's milk. RAST and immunoblotting were also performed. RESULTS: Results of skin testing and RAST with cow's milk were negative but demonstrated an IgE-mediated allergy to mare's milk. Immunoblotting revealed two allergen bands with molecular weights of 16 and 18 kd, most likely representing the whey proteins alpha-lactalbumin and beta-lactoglobulin. The bands disappeared after the mare's milk was boiled, indicating that the proteins are heat-labile. CONCLUSION: The results of this study demonstrate the existence of an IgE-mediated mare's milk allergy caused by low molecular weight heat-labile proteins, most likely alpha-lactalbumin and beta-lactoglobulin, which do not cross-react with the corresponding whey proteins in cow's milk.