Structure and dynamics of the alpha-lactalbumin molten globule: fluorescence studies using proteins containing a single tryptophan residue

The fluorescence properties of three variants of alpha-lactalbumin (alpha-LA) containing a single tryptophan residue were investigated under native, molten globule, and unfolded conditions. These proteins have levels of secondary structure and stability similar to those of the wild type. The fluorescence signal in the native state is dominated by that of W104, with the signal of W60 and W118 significantly quenched by the disulfide bonds in their vicinity. In the molten globule state, the magnitude of the fluorescence signal of W60 and W118 increases, due to the loss of rigid, specific side chain packing. In contrast, the magnitude of the signal of W104 decreases in the molten globule state, perhaps due to the protonation of H107 or quenching by D102 or K108. The solvent accessibilities of individual tryptophan residues were investigated by their fluorescence emission maximum and by acrylamide quenching studies. In the native state, the order of solvent accessibility is as follows: W118 > W60 > W104. This order changes to W60 > W104 > W118 in the molten globule state. Remarkably, the solvent accessibility of W118 in the alpha-LA molten globule is lower than that in the native state. The dynamic properties of the three tryptophan residues were examined by time-resolved fluorescence anisotropy decay studies. The overall rotation of the molecule can be observed in both the native and molten globule states. In the molten globule state, there is an increase in the extent of local backbone fluctuations with respect to the native state. However, the fluctuation is not sufficient to result in complete motional averaging. The three tryptophan residues in the native and molten globule states have different degrees of motional freedom, reflecting the folding pattern and dynamic heterogeneity of these states. Taken together, these studies provide new insight into the structure and dynamics of the alpha-LA molten globule, which serves as a prototype for partially folded proteins.     

Exploration of partially unfolded states of human alpha-lactalbumin by molecular dynamics simulation

Molecular dynamics simulations are used to probe the properties of non-native states of the protein human alpha-lactalbumin (human alpha-LA) with a detailed atomistic model in an implicit aqueous solvent environment. To sample the conformational space, a biasing force is introduced that increases the radius of gyration relative to the native state and generates a large number of low-energy conformers that differ in terms of their root-mean-square deviation, for a given radius of gyration. The resulting structures are relaxed by unbiased simulations and used as models of the molten globule and partly denatured states of human alpha-LA, based on measured radii of gyration obtained from nuclear magnetic resonance experiments. The ensembles of structures agree in their overall properties with experimental data available for the human alpha-LA molten globule and its more denatured states. In particular, the simulation results show that the native-like fold of the alpha-domain is preserved in the molten globule. Further, a considerable proportion of the antiparallel beta-strand in the beta-domain are present. This indicates that the lack of hydrogen exchange protection found experimentally for the beta-domain is due to rearrangement of the beta-sheet involving transient populations of non-native beta-structures. The simulations also provide details concerning the ensemble of structures that contribute as the molten globule unfolds and shows, in accord with experimental data, that unfolding is not cooperative; i.e. the various structural elements do not unfold simultaneously.     

Stepwise proteolytic removal of the beta subdomain in alpha-lactalbumin. The protein remains folded and can form the molten globule in acid solution.

Bovine alpha-lactalbumin (alpha-LA) is an alpha/beta protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of alpha-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the beta subdomain is less structured. Here, we investigate the conformational features of three derivatives of alpha-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the beta subdomain of the native protein (approximately from residue 34 to 57). These alpha-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked alpha-LA species consisting of fragments 1-,3-40 and 41-123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of alpha-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1-40 and 53-123 (desbeta1-LA) or fragments 1-34 and 54-,57-123 (desbeta2-LA) were obtained by digestion of alpha-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped alpha-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact alpha-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the alpha-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three alpha-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact alpha-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the beta subdomain can be removed from the alpha-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the alpha-LA molten globule only the alpha domain is structured.     

Stability of the molten globule state of a domain-exchanged chimeric protein between human and bovine alpha-lactalbumins

A domain-exchanged chimeric alpha-lactalbumin (alpha-LA), which consisted of the alpha-domain of human alpha-LA and the beta-domain of bovine alpha-LA, was constructed. Like native alpha-LA, the chimeric protein was in a molten globule state in the absence of Ca(2+) at neutral pH and low salt concentration. The stability of the molten globule state of the constructed chimeric protein was identical to that of the recombinant human protein and was higher than that of the recombinant bovine protein. The stability of the molten globule state of alpha-LA is defined by the stability of the alpha-domain.     

Local and global cooperativity in the human alpha-lactalbumin molten globule

NMR spectroscopy has been used to follow the urea-induced unfolding of the low pH molten globule states of a single-disulfide variant of human alpha-lactalbumin ([28-111] alpha-LA) and of two mutants, each with a single proline substitution in a helix. [28-111] alpha-LA forms a molten globule very similar to that formed by the wild-type four-disulfide protein, and this variant has been used as a model for the alpha-lactalbumin (alpha-LA) molten globule in a number of studies. The urea-induced unfolding behavior of [28-111] alpha-LA is similar to that of the four-disulfide form of the protein, except that [28-111] alpha-LA is less stable and has greater cooperativity in the loss of different elements of structure. For one mutant, L11P, the helix containing the mutation is highly destabilized such that it is completely unfolded even in the absence of urea. By contrast, for the other mutant, Q117P, the helix containing the mutation retains its compact structure. Both mutations, however, show significant long-range destabilization of the overall fold showing that the molten globule state has a degree of global cooperativity. The results reveal that different permutations of three of the four major alpha-helices of the protein can form a stable, locally cooperative, compact structural core. Taken together, these findings demonstrate that the molten globule state of alpha-LA is an ensemble of conformations, with different subsets of structures linked by a range of long-range interactions.     

Electrostatic interactions in the acid denaturation of alpha-lactalbumin determined by NMR.

alpha-Lactalbumin (alpha-LA) undergoes a pH-dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pig alpha-LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation in alpha-LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation of alpha-LA are concentrated in the calcium binding region of the protein.     

A model of dynamic side-chain--side-chain interactions in the alpha-lactalbumin molten globule

Proteins in the molten globule state contain high levels of secondary structure, as well as a rudimentary, nativelike tertiary topology. Thus, the structural similarity between the molten globule and native proteins may have a significant bearing in understanding the protein-folding problem. To explore the nature of side-chain--side-chain interactions in the alpha-lactalbumin (alpha-LA) molten globule, we determined the effective concentration for formation of the 28--111 disulfide bond in 14 double-mutant proteins, each containing two hydrophobic core residues replaced by alanine. We compared our results with those of single-alanine substitutions using the framework of double-mutant cycle analysis and found that, in the majority of cases, the effects of two alanine substitutions are additive. Based on these results, we propose a model of side-chain-side-chain interactions in the alpha-LA molten globule, which takes into consideration the dynamic nature of this partially folded species.     

The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin

The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA.     

Alpha-lactalbumin forms a compact molten globule in the absence of disulfide bonds.

Human alpha-lactalbumin (alpha-LA) is a four disulfide-bonded protein that adopts partially structured conformations under a variety of mildly denaturing conditions. At low pH, the protein is denatured but compact, with a high degree of secondary structure and a native-like fold. This is commonly referred to as a molten globule. A variant of alpha-LA, in which all eight cysteines have been mutated to alanine (all-Ala alpha-LA), has been studied using NMR spectroscopy. At low pH all-Ala alpha-LA is nearly as compact as wild type alpha-LA. Urea-induced unfolding experiments reveal that the residues that remain compact in the absence of disulfide bonds are those that are most resistant to unfolding in the wild-type alpha-LA molten globule. This is particularly remarkable because this stable core is formed by segments of the polypeptide chain from both the N- and C-termini. These results show that the overall architecture of the protein fold of alpha-LA is determined by the polypeptide sequence itself, and not as the result of cross-linking by disulfide bonds, and provide insight into the way in which the sequence codes for the fold.     

Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein

Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein.     

A stabilized molten globule protein

A predominant conformational isomer of non-native alpha-lactalbumin (alpha-LA) has been purified by thermal denaturation of the native alpha-LA using the technique of disulfide scrambling. This unique isomer retains a substantial content of alpha-helical structure. It is stabilized by two native disulfide bonds within the alpha-helical domain and two scrambled non-native disulfide bonds at the beta-sheet domain. This denatured isomer of alpha-LA exhibits structural characteristics that are consistent with the well-documented molten globule state. The ability to prepare a stabilized and structurally defined molten globule provides a useful model for studying the folding and unfolding pathways of proteins.     

Characterization of the molten globule of human serum retinol-binding protein using NMR spectroscopy

The molten globule state is a partially folded conformer of proteins that has been the focus of intense study for more than two decades. This non-native fluctuating conformation has been linked to protein-folding intermediates, to biological function, and more recently to precursors in amyloid fibril formation. The molten globule state of human serum retinol-binding protein (RBP) has been postulated previously to be involved in the mechanism of ligand release (Ptitsyn, O. B., et al. (1993) FEBS Lett. 317, 181-184). Conserved residues within RBP have been identified and proposed to be key to folding and stability, although a link to a molten globule state has not previously been shown (Greene, L. H., et al. (2003) FEBS Lett. 553, 39-44). In this work, a detailed characterization of the acid-induced molten globule of RBP is presented. Using stopped-flow fluorescence spectroscopy in the presence of 8-anilino-1-naphthalene sulfonic acid (ANS), we show that RBP populates a state with molten-globule-like characteristics early in refolding. To gain insight into the structural features of the molten globule of RBP, we have monitored the denaturant-induced unfolding of this ensemble using NMR spectroscopy. The transition at the level of individual residues is significantly more cooperative than that found previously for the archetypal molten globule, alpha-lactalbumin (alpha-LA); this difference may be due to a predominantly beta-sheet structure present in RBP in contrast to the alpha-helical nature of the alpha-LA molten globule.     

Organization and dynamics of tryptophans in the molten globule state of bovine α-lactalbumin utilizing wavelength-selective fluorescence approach: Comparisons with native and denatured states

Bovine α-lactalbumin (BLA) is known to be present in molten globule form in its apo-state (i.e., Ca²⁺ depleted state). We explored the organization and dynamics of the functionally important tryptophan residues of BLA in native, molten globule and denatured states utilizing the wavelength-selective fluorescence approach. We observed red edge excitation shift (REES) of 7 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit considerable REES (8 nm) in its molten globule state. Taken together, these results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment. We further show that even the denatured form of BLA exhibits a modest REES of 3 nm, indicating that the tryptophans are shielded from bulk solvent, even when denatured, due to the presence of residual structure around tryptophan(s). This is further supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These novel results constitute one of the first reports of REES in the molten globule state of proteins, and could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.     

Nanosecond dynamics of tryptophans in different conformational states of apomyoglobin proteins

Fluorescence anisotropy kinetics were employed to quantify the nanosecond mobility of tryptophan residues in different conformational states (native, molten globule, unfolded) of apomyoglobins. Of particular interest is the similarity between the fluorescence anisotropy decays of tryptophans in the native and molten globule states. We find that, in these compact states, tryptophan residues rotate rapidly within a cone of semiangle 22-25 degrees and a correlation time of 0.5 ns, in addition to rotating together with the whole protein with a correlation time of 7-11 ns. The similar nanosecond dynamics of tryptophan residues in both states suggests that the conformation changes that distinguish the molten globule and native states of apomyoglobins originate from either subtle, slow rearrangements or fast changes distant from these tryptophans.     

Molecular dynamics model structures for the molten globule state of alpha-lactalbumin: aromatic residue clusters I and II

To model the molten globule structure of alpha-lactalbumin, molecular dynamics (MD) simulations were carried out for the protein in explicit water at high temperature. In these simulations, long-range Coulomb interactions were evaluated explicitly with an original method (particle-particle and particle-cell: PPPC) to avoid artifacts caused by the cut-off. The MD simulations were started from two initial conditions to verify that similar results would be obtained. From the last 150 ps trajectories of the two MD simulations, two partially unfolded average structures were obtained. These structures had the following common structural features which are characteristic of the molten globule state. The radii of gyration for these conformations were 7.4 and 9.6% larger than that of the native state. These values were almost the same as the experimental value (9.6%) observed recently by small-angle X-ray scattering (Kataoka,M., Kuwajima,K., Tokunaga,F. and Goto,Y., 1997, Protein Sci., 6, 422-430). Furthermore, aromatic residues of clusters I and II in these structures were far apart from each other except for Try103-Trp104. This result is in good agreement with NMR experimental results for the acid-denatured molten globule state (Alexandrescu et al., 1992, 1993); that is, NOE signals between the aromatic residues were not observed, except for that of Try103-Trp104 in the molten globule state. Other structural features of these models for the molten globule state are discussed with reference to native state structures.     

Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of DeltaCp of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.     

Exploring tryptophan dynamics in acid-induced molten globule state of bovine α-lactalbumin: a wavelength-selective fluorescence approach

The relevance of partially ordered states of proteins (such as the molten globule state) in cellular processes is beginning to be understood. Bovine α-lactalbumin (BLA) assumes the molten globule state at acidic pH. We monitored the organization and dynamics of the functionally important tryptophan residues of BLA in native and molten globule states utilizing the wavelength-selective fluorescence approach and fluorescence quenching. Quenching of BLA tryptophan fluorescence using quenchers of varying polarity (acrylamide and trichloroethanol) reveals varying degrees of accessibility of tryptophan residues, characteristic of native and molten globule states. We observed red edge excitation shift (REES) of 6 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit REES (3 nm) in the molten globule state. These results constitute one of the early reports of REES in the molten globule state of proteins. Taken together, our results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment and that the regions surrounding at least some of the BLA tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited-state tryptophans. These results are supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These results could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.     

Structural characterisation of the human alpha-lactalbumin molten globule at high temperature

Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 degrees C leads to a dramatic sharpening of peaks in the 15N-1H HSQC spectrum of human alpha-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human alpha-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the H(alpha), H(N) and 15N chemical shifts of the human alpha-LA molten globule at 50 degrees C leads to the identification of regions of native-like helix in the alpha-domain and of non-native helical propensity in the beta-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments.