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.     

Characterization of millisecond time-scale dynamics in the molten globule state of alpha-lactalbumin by NMR

The motional dynamics of the molten globule (MG) state of alpha-lactalbumin have been characterized using (15)N transverse relaxation rates (R2). A modified version of the Carr-Purcell-Meiboom-Gill (CPMG) R2 pulse sequence is proposed in order to overcome the loss of sensitivity that arises from extreme line broadening due to complex dynamics on the millisecond time-scale. Using this pulse sequence, chemical exchange rates were extracted by examining the (15)N transverse relaxation rates as a function of CPMG delay values. The results clearly illustrate that pervasive conformational exchange of 0.2-0.5 ms in the (15)N backbone resonances of the molten globule state of alpha-lactalbumin. The temperature dependence of the conformational exchange rates display standard Arrhenius kinetic behavior between 10 and 30 degrees C. Estimates of the activation energies range from 0.8 to 4. 4 kcal/mol, indicating a low energetic barrier to conformational fluctuations relative to native state proteins. The fluctuations and low energetic barriers may be critical for directing the search for contacts that will result in the transition from the MG state to the native state.     

Pressure-induced unfolding of the molten globule of all-Ala alpha-lactalbumin

Pressure-induced unfolding of a molten globule (MG) was studied in a residue-specific manner with (1)H-(15)N two-dimensional NMR spectroscopy using a variant of human alpha-lactalbumin (alpha-LA), in which all eight cysteines had been replaced with alanines (all-Ala alpha-LA). The NMR spectrum underwent a series of changes from 30 to 2000 bar at 20 degrees C and from -18 degrees C to 36 degrees C at 2000 bar, showing a highly heterogeneous unfolding pattern according to the secondary structural elements of the native structure. Unfolding began in the loop part of the beta-domain, and then extended to the remainder of the beta-domain, after which the alpha-domain began to unfold. Within the alpha-domain, the pressure stability decreased in the order: D-helix approximately 3(10)-helix > C-helix approximately B-helix > A-helix. The D-helix, C-terminal 3(10)-helix and a large part of B- and C-helices did not unfold at 2000 bar, even at 36 degrees C or at -18 degrees C. The results verify that the MG state consists of a mixture of variously unfolded conformers from the mostly folded to the nearly totally unfolded that differ in stability and partial molar volume. Not only heat but also cold denaturation was observed, supporting the view that the MG state is stabilized by hydrophobic interactions.     

Entropy calculations on the molten globule state of a protein: side-chain entropies of alpha-lactalbumin.

We present entropy estimates based on molecular dynamics simulations of models of the molten globule state of the protein alpha-lactalbumin at low pH. The entropy calculations use the covariance matrix of atom-positional fluctuations and yield the complete configurational entropy. The configurational entropy of the entire protein and of each of its side chains is calculated. Exposed side chains show a larger entropy compared to buried side chains. A comparison to data from rotamer counting is made and significant differences are found.     

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.     

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.     

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.     

Effect of hydrostatic pressure on unfolding of alpha-lactalbumin: volumetric equivalence of the molten globule and unfolded state

The effect of pressure on the unfolding of bovine alpha-lactalbumin was investigated by ultraviolet absorption methods. The change of molar volume associated with unfolding, deltaV, was measured in the presence or absence of guanidine hydrochloride at pH 7. The deltaV was estimated to be -63 cm3/mol in the absence of a chemical denaturant. While in the presence of guanidine hydrochloride (GuHCl), it was found that deltaV was -66 cm3/mol at 25 degrees C and was independent of the concentration of GuHCl, despite the fact that the molten globule fraction in the total unfolding product decreased with the increase of GuHCl concentration. The results indicate that the volume of alpha-lactalbumin only changes at the transition from a native to a molten globule state, and almost no volume change has been found during the transition from a molten globule to the unfolded state.     

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.     

1,1,1,3,3,3-hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine alpha-lactalbumin: calorimetric and spectroscopic studies

The thermal denaturation of alpha-lactalbumin was studied at pH 7.0 and 9.0 in aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) by high-sensitivity differential scanning calorimetry. The conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. The most obvious effect of HFIP was lowering of the transition temperature with an increase in the concentration of the alcohol up to 0.30M, beyond which no calorimetric transition was observed. Up to 0.30M HFIP the calorimetric and van't Hoff enthalpy remained the same, indicating the validity of the two-state approximation for the thermal unfolding of alpha-lactalbumin. The quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. Spectroscopic observations confirm that alpha-lactalbumin is in the molten globule state in the presence of 0.50M HFIP at pH 7.0 and 0.75M HFIP at pH 9.0. The results also demonstrate that alpha-lactalbumin in the molten globule state undergoes a noncooperative thermal transition to the denatured state. It is observed that two of four tryptophans are exposed to the solvent in the HFIP induced molten globule state of alpha-lactalbumin compared to four in the 8.5M urea induced denatured state of the protein. It is also observed that the HFIP induced molten globule states at the two pH values are different from the acid induced molten globule state (A state) of alpha-lactalbumin.     

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.     

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.     

Local and long-range interactions in the molten globule state: A study of chimeric proteins of bovine and human alpha-lactalbumin

The molten globule state of alpha-lactalbumin has ordered secondary structure in the alpha-domain, which comprises residues 1 to 34 and 86 to 123. In order to investigate which part of a polypeptide is important for stabilizing the molten globule state of alpha-lactalbumin, we have produced and studied three chimeric proteins of bovine and human alpha-lactalbumin. The stability of the molten globule state formed by domain-exchanged alpha-lactalbumin, in which the amino acid sequence in the alpha-domain comes from human alpha-lactalbumin and that in the beta-domain comes from bovine alpha-lactalbumin, is the same as that of human alpha-lactalbumin and is substantially greater than that of bovine alpha-lactalbumin. Therefore, our results show that the stability of the molten globule state of alpha-lactalbumin is determined by the alpha-domain and the beta-domain is not important for stabilizing the molten globule state. The substitution of residues 1 to 34 of bovine alpha-lactalbumin with those of human alpha-lactalbumin substantially increases the stability of the molten globule state, while the substitution of residues 86 to 123 of bovine alpha-lactalbumin with those of human alpha-lactalbumin decreases the stability of the molten globule state. Therefore, residues 1 to 34 in human alpha-lactalbumin is more important for the stability of the human alpha-lactalbumin molten globule state than residues 86 to 123. The stabilization of the molten globule state due to substitution of both residues 1 to 34 and 86 to 123 is not identical with the sum of the two individual substitutions, demonstrating the non-additivity of the stabilization of the molten globule state. This result indicates that there is a long-range interaction between residues 1 to 34 and 86 to 123 in the molten globule state of human alpha-lactalbumin. The differences in the stabilities of the molten globule states are well correlated with the averaged helical propensity values in the alpha-domain when the long-range interactions are negligible, suggesting that the local interaction is the dominant term for determining the stability of the molten globule state. Our results also indicate that the apparent cooperativity is closely linked to the stability of the molten globule state, even if the molten globule state is weakly cooperative.     

Effects of the stabilization of the molten globule state on the folding mechanism of alpha-lactalbumin: a study of a chimera of bovine and human alpha-lactalbumin

The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.     

pH-dependent stability of the human alpha-lactalbumin molten globule state: contrasting roles of the 6 - 120 disulfide and the beta-subdomain at low and neutral pH

Many proteins are capable of populating partially folded states known as molten globule states. Alpha-lactalbumin forms a molten globule under a range of conditions including low pH (the A-state) and at neutral pH in the absence of Ca(2+) with modest amounts of denaturant. The A-state is the most thoroughly characterized and thought to mimic a kinetic intermediate populated during refolding at neutral pH. We demonstrate that the properties and interactions that stabilize the A-state and the pH 7 molten globule of human alpha-lactalbumin differ. The unfolding of the wild-type protein is compared to the unfolding of a variant that lacks the 6 - 120 disulfide bond and to an autonomously folded peptide construct that we have previously shown represents the minimum core structure of the A-state of human alpha-lactalbumin. Studies conducted at pH 2 and 7 show that the disulfide makes little contribution to the stability of the molten globule at pH 7 but is important at pH 2. In contrast, the beta-subdomain of the protein is less important at pH 2 than at pH 7. The role of helix propensity in stabilizing the different forms of the molten globule state is examined and it is shown that it cannot account for the differences. The strikingly different behavior observed at pH 2 and 7 indicates that the A-state may not be a rigorous mimic of the folding intermediate populated at pH 7.     

Structural energetics of the molten globule state

Certain partly ordered protein conformations, commonly called “moltenglobule states,” are widely believed to represent protein folding intermediates. Recentstructural studies of molten globule states ofdifferent proteins have revealed features whichappear to be general in scope. The emergingconsensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define ageneral state of protein folding called “the molten globule state,” which is structurally andthermodynamically distinct from both the native state and the denatured state. Despite exaatensive knowledge of structural features of afew molten globule states, a cogent thermodynamic argument for their stability has not yetbeen advanced. The prevailing opinion of thelast decade was that there is little or no enthalpy difference or heat capacity differencebetween the molten globule state and the unfolded state. This view, however, appears to beat variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of α-lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other protential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states. © 1993 Wiley-Liss, Inc.     

Pressure-induced molten globule state of cholinesterase

The denaturing effect of pressure on the structure of human butyrylcholinesterase was examined by gel electrophoresis under pressure and by 8-anilino-1-naphthalene sulfonate (ANS) binding. It was found that the fluorescence intensity of bound ANS is increased by pressure between 0.5 and 1.5 kbar and that the hydrodynamic volume of the enzyme swells when pressures around 1.5 kbar are applied. These findings indicate that pressure denaturation of butyrylcholinesterase is a multi-step process and that the observed transient pressure-denatured states have characteristics of molten globules.     

Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig alpha-lactalbumin

NMR spectroscopy has been used to investigate the structure of a partially folded state of a protein, the molten globule or A-state of alpha-lactalbumin. The 1H NMR spectrum of this species differs substantially from those of both the native and fully unfolded states, reflecting the intermediate level of order. The resolution in the spectrum is limited by the widespread overlap and substantial line widths of many of the resonances. Methods have therefore been developed that exploit the well-resolved spectrum of the native protein to probe indirectly the A-state. A number of resonances of the A-state have been found to be substantially shifted from their positions in the spectrum of the unfolded state and have been identified through magnetization transfer with the native state, under conditions where the two states are interconverting. The most strongly perturbed residues in the A-state were found to be among those that form a hydrophobic core to the native structure. A number of amides were found to be highly protected from solvent exchange in the A-state. These have been identified through pH-jump experiments, which label them in the spectrum of the native protein. They were found to occur mainly in segments that are helical in the native structure. These results enable a model of the A-state to be proposed in which significant conformational freedom exists but where specific elements of native-like structure are preserved.     

Increased exposure of hydrophobic surface in molten globule state of alpha-lactalbumin. Fluorescence and hydrophobic photolabeling studies.

The involvement of molten globule state as a distinct intermediate in the denaturation process in proteins is well documented. However, the structural characterization of such an intermediate is far from complete. We have, using fluorescence and fluorescence quenching, studied the molten globule state of bovine alpha-lactalbumin. Unlike the native state, where all the 4 tryptophans are buried in the protein, 2 tryptophans are exposed in the molten globule state. Using the hydrophobic photoactivable reagent [3H]diazofluorene, we observe an increased hydrophobic exposure in the molten globule state. These structural characteristics conform to the current views on the molten globule state, i.e. it has similar secondary structure but a poorly defined tertiary structure. Our fluorescence studies indicate the involvement of a premolten globule state in the native to molten globule state transition. This premolten globule state exists at pH 5.0 and has a very compact structure involving increased hydrophobic interactions in the protein interior. These results are also supported by circular dichroism studies.     

Thermodynamic and structural properties of the acid molten globule state of horse cytochrome C

To understand the stabilization, folding, and functional mechanisms of proteins, it is very important to understand the structural and thermodynamic properties of the molten globule state. In this study, the global structure of the acid molten globule state, which we call MG1, of horse cytochrome c at low pH and high salt concentrations was evaluated by solution X-ray scattering (SXS), dynamic light scattering, and circular dichroism measurements. MG1 was globular and slightly (3%) larger than the native state, N. Calorimetric methods, such as differential scanning calorimetry and isothermal acid-titration calorimetry, were used to evaluate the thermodynamic parameters in the transitions of N to MG1 and MG1 to denatured state D of horse cytochrome c. The heat capacity change, ΔC(p), in the N-to-MG1 transition was determined to be 2.56 kJ K(-1) mol(-1), indicating the increase in the level of hydration in the MG1 state. Moreover, the intermediate state on the thermal N-to-D transition of horse cytochrome c at pH 4 under low-salt conditions showed the same structural and thermodynamic properties of the MG1 state in both SXS and calorimetric measurements. The Gibbs free energy changes (ΔG) for the N-to-MG1 and N-to-D transitions at 15 °C were 10.9 and 42.2 kJ mol(-1), respectively.