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.     

Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering.

A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the alpha-lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of alpha-lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of alpha-lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 A for the native state to 60 A for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the alpha-lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the alpha-lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.     

Spontaneous asparaginyl deamidation of canine milk lysozyme under mild conditions

Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.     

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.     

Defining the core structure of the alpha-lactalbumin molten globule state.

Molten globules are partially folded states of proteins which are generally believed to mimic structures formed during the folding process. In order to determine the minimal requirements for the formation of a molten globule state, we have prepared a set of peptide models of the molten globule state of human alpha-lactalbumin (alphaLA). A peptide consisting of residues 1-38 crosslinked, via the native 28-111 disulfide bond, to a peptide corresponding to residues 95-120 forms a partially folded state at pH 2.8 which has all of the characteristics of the molten globule state of alphaLA as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation experiments. The structure of the peptide construct is the same at pH 7.0. Deletion of residues 95-100 from the construct has little effect. Thus, less than half the sequence is required to form a molten globule. Further truncation corresponding to the selective deletion of the A (residues 1-19) or D (residues 101-110) helices or the C-terminal 310 helix (residues 112-120) leads to a significant loss of structure. The loss of structure which results from the deletion of any of these three regions is much greater than that which would be expected based upon the non-cooperative loss of local helical structure. Deletion of residues corresponding to the region of the D helix or C-terminal 310 helix region results in a peptide construct which is largely unfolded and contains no more helical structure than is expected from the sum of the helicity of the two reduced peptides. These experiments have defined the minimum core structure of the alphaLA molten globule state.     

Side chain accessibility and dynamics in the molten globule state of alpha-lactalbumin: a (19)F-NMR study

The molten globule state of alpha-lactalbumin (alpha-LA) has been considered a prototype of partially folded proteins. Despite the importance of molten globules in understanding the mechanisms of protein folding and its relevance to some biological phenomena, site-specific information on the structure and dynamics of a molten globule is limited, largely because of the high conformational flexibility and heterogeneity. Here, we use selective isotope labeling and (19)F NMR to investigate the solvent accessibility and side-chain dynamics of aromatic residues in the molten globule of alpha-LA. Comparison of these properties with those of the native and unfolded protein indicates that the alpha-LA molten globule is highly heterogeneous; each residue has its unique solvent accessibility and motional environment. Many aromatic residues normally buried in the interior of native alpha-LA remain significantly buried in the molten globule and the side-chain dynamics of these residues are highly restricted. Our results suggest that hydrophobic and van der Waals interactions mediated by the inaccessible surface area could be sufficient to account for all the stability of the alpha-LA molten globule, which is approximately 50% of the value for the native protein.     

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.     

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.     

A protein dissection study demonstrates that two specific hydrophobic clusters play a key role in stabilizing the core structure of the molten globule state of human alpha-lactalbumin

The molten globule state of alpha-lactalbumin (alpha LA) has served as a paradigm for understanding the role of these partially folded states in protein folding. We previously showed that a peptide construct consisting of the A and B helices (residues 1-38) cross-linked to the D- and C-terminal 3(10) helices (residues 101-120) of alpha LA is capable of folding to a stable molten globule-like state. Here, we report the study of three peptide constructs that are designed to investigate the contribution two short hydrophobic sequences located near the C-terminus of alpha LA make to the structure and stability of the alpha LA molten globule state. These regions of the protein have been shown to form stable non-native structures in isolation. The three peptide constructs contain residues 1-38 cross-linked to three separate C-terminal peptides via the native 28-111 disulfide bond. The C-terminal peptides consist of residues 101-114, 106-120, and 106-114. The results of CD, fluorescence, ANS binding, and urea denaturation experiments indicate that constructs that lack either of the hydrophobic sequences (residues 101-105 and 115-120) are significantly less structured. These results highlight the importance of long-range, mutually stabilizing interactions within the molten globule state of the protein. Proteins 2001;42:237-242.     

Molten globule formation in apomyoglobin monitored by the fluorescent probe Nile Red

The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data.     

Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach

Retinol-binding protein transports retinol, and circulates in the plasma as a macromolecular complex with the protein transthyretin. Under acidic conditions retinol-binding protein undergoes a transition to the molten globule state, and releases the bound retinol ligand. A biased molecular dynamics simulation method has been used to generate models for the ensemble of conformers populated within this molten globule state. Simulation conformers, with a radius of gyration at least 1.1 A greater than that of the native state, contain on average 37%beta-sheet secondary structure. In these conformers the central regions of the two orthogonal beta-sheets that make up the beta-barrel in the native protein are highly persistent. However, there are sizable fluctuations for residues in the outer regions of the beta-sheets, and large variations in side chain packing even in the protein core. Significant conformational changes are seen in the simulation conformers for residues 85-104 (beta-strands E and F and the E-F loop). These changes give an opening of the retinol-binding site. Comparisons with experimental data suggest that the unfolding in this region may provide a mechanism by which the complex of retinol-binding protein and transthyretin dissociates, and retinol is released at the cell surface.     

Enhanced susceptibility to transglutaminase reaction of α-lactalbumin in the molten globule state

The susceptibility of a-lactalbumin to transglutaminase reactions was studied using an enzyme from Streptoverticillium which can catalyze the reactions irrespective of the presence or absence of Ca²⁺. Transglutaminase-catalyzed polymerization of a-lactalbumin in the native state occurred to a very limited extent. Transformation from the native state to the molten globule state brought about by Ca²⁺-removal from holo-a-lactalbumin enhanced the polymerization of the protein catalyzed by transglutaminase. The incorporation of Carbobenzoxy-Gln-Gly into a-lactalbumin through the enzyme reaction was investigated to determine the amounts of lysine residues which are present at molecular surface and available to the enzyme. There was no significant difference in the amount of available lysine residues between the native: and the molten globule molecule. However, the amount of surface glutamine residues incorporated with monodansylcadaverine by transglutaminase was remarkably higher in the molten globule state than that in the native state. The monodansylcadaverine-incorporated site of a-lactalbumin in the molten globule state was identified as Gln-54 by amino-acid sequence analysis of fluorescence-labeled peptides separated from chymotryptic digests of the protein. Possible reason for selective labeling of Gln-54 in molten globule a-lactalbumin was proposed.     

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.     

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.     

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.     

Calorimetric determination of the energetics of the molten globule intermediate in protein folding: apo-alpha-lactalbumin

High-sensitivity differential scanning calorimetry has been used to characterize the energetics of the molten globule state of apo-alpha-lactalbumin. This characterization has been possible by performing temperature scans at different guanidine hydrochloride (GuHCl) concentrations in order to experimentally define the temperature-GuHCl stability surface of the protein. Multidimensional analysis of the heat capacity surface has allowed simultaneous resolution of the energetics of the unfolded and molten globule states. These experiments indicate that the intrinsic enthalpy difference (i.e., excluding additional contributions such as those arising from differential GuHCl binding) between the unfolded and native states is 31.8 kcal/mol at 25 degrees C whereas that of the molten globule and native states is only 7.7 kcal/mol. At the same temperature, the entropy changes are 99.2 and 23.7 cal/K.mol and the heat capacity changes are 1821 and 326 cal/K.mol, respectively. Analysis of the thermodynamic data indicates that in passing from the native to the molten globule state only approximately 19% of the hydrogen bonds are broken. In addition, the magnitude of delta Cp for the molten globule suggests that water does not largely penetrate into the interior of the molten globule, implying that significant hydrophobic interactions are still present in this state. These parameters provide precise energetic constraints to the allowed structural conformations of the molten globule.     

The molten globule state of a chimera of human alpha-lactalbumin and equine lysozyme.

The molten globule state of equine lysozyme is more stable than that of alpha-lactalbumin and is stabilized by non-specific hydrophobic interactions and native-like hydrophobic interactions. We constructed a chimeric protein which is produced by replacing the flexible loop (residues 105-110) in human alpha-lactalbumin with the helix D (residues 109-114) in equine lysozyme to investigate the possible role of the helix D for the high stability and native-like packing interaction in the molten globule state of equine lysozyme. The stability of the molten globule state formed by the chimeric protein to guanidine hydrochloride-induced unfolding is the same as that of equine lysozyme and is substantially greater than that of human alpha-lactalbumin, although only six residues come from equine lysozyme. Our results also suggest that the non-native interaction in the molten globule state of alpha-lactalbumin changes to the native-like packing interaction due to helix substitution. The solvent-accessibility of the Trp residues in the molten globule state of the chimeric protein is similar to that in the molten globule state of equine lysozyme in which packing interaction around the Trp residues in the native state is partially preserved. Therefore, the helix D in equine lysozyme is one of the contributing factors to the high stability and native-like packing interaction in the molten globule state of equine lysozyme. Our results indicate that the native-like packing interaction can stabilize the rudimentary intermediate which is stabilized by the non-specific hydrophobic interactions.     

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.     

Characterization of acid-induced unfolding intermediates of glucose/xylose isomerase.

Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.