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

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

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.     

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.     

Probing subtle differences in the hydrogen exchange behavior of variants of the human alpha-lactalbumin molten globule using mass spectrometry

The hydrogen-exchange behavior of the low-pH molten globule of human alpha-lactalbumin, containing all four disulfides, has been examined and compared with that of a single disulfide variant, [28-111] alpha-lactalbumin, and of a series of proline variants of [28-111] alpha-lactalbumin. The small differences in hydrogen-exchange protection exhibited by these partially folded species were compared by mixing two or more proteins and monitoring their exchange simultaneously using mass spectrometry. The effect of single proline mutations within each alpha-domain helix on hydrogen-exchange protection has been investigated using six proline variants of [28-111] alpha-lactalbumin, L11P, L12P, M30P, I95P, K108P and Q117P. The results show that proline mutations in the A, B, C and D alpha-helices lead to a loss of hydrogen-exchange protection for residues in the local helix without perturbing hydrogen-exchange protection in other regions of the protein. Thus, local unfolding of the A, B, C and D helices does not significantly alter the packing and solvent accessibility of other regions of the molten globule. By contrast, introduction of a proline residue in the C-terminal 3(10) helix produces a larger and more widespread loss of hydrogen-exchange protection, demonstrating that longer-range perturbations of the molten globule have occurred. Thus, residues in this C-terminal region must be involved in contacts that are critical for the stabilisation of the compact molten globule structure.     

Hydrogen exchange study of canine milk lysozyme: stabilization mechanism of the molten globule

The native state (1)H, (15)N resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and alpha-lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two-state model, whereas that of alpha-lactalbumin follows a three-state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of alpha-lactalbumin but with extreme stability. Study of the molten globules of alpha-lactalbumin and equine milk lysozyme showed that the stabilities of their alpha-helices are similar, despite the differences in the thermodynamic stability of their molten globule states. On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the alpha-helices are more stabilized than in alpha-lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state.     

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

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

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.     

Cold denaturation of alpha-lactalbumin

We have investigated the thermal unfolding of bovine alpha-lactalbumin by means of circular dichroism spectroscopy in the far- and near-ultraviolet regions, and shown that the native alpha-lactalbumin undergoes heat and cold denaturation. The guanidine hydrochloride-induced unfolding of alpha-lactalbumin was also investigated by circular dichroism spectroscopy at various temperatures from 261 to 318 K. It is shown that the population of the molten globule state is strongly dependent on temperature and that the molten globule state does not accumulate during the guanidine hydrochloride-induced unfolding transition at 261 K. Our results indicate that the molten globule state of alpha-lactalbumin undergoes cold denaturation as the native alpha-lactalbumin does, and that the heat capacity change of unfolding from the molten globule to the unfolded state is positive and significant. The present results further support the idea that the molten globule and the unfolded states do not belong to the same thermodynamic state, and that the native, molten globule and unfolded states are sufficient for interpreting the guanidine hydrochloride-induced unfolding behavior of alpha-lactalbumin.     

Spin diffusion in globular proteins: alpha-lactalbumin

The structural properties of globular proteins analyzed by two different methods: high-resolution NMR and circular dichroism were compared. We established that the spin diffusion method shows changes in the secondary structure during the unfolding of the alpha-lactalbumin molten globule by urea. It was shown that the spin diffusion method is extremely effective in studies of interactions of water and denaturant molecules with the protein both in the native and the molten globule states.     

Equilibrium and kinetics of the folding and unfolding of canine milk lysozyme

The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.     

Energetic basis of structural stability in the molten globule state: alpha-lactalbumin

The denatured states of alpha-lactalbumin, which have features of a molten globule state, have been studied to elucidate the energetics of the molten globule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity increment of alpha-lactalbumin denaturation highly correlates with the degree of disorder of the residual structure of the state. As a result, the denaturational transition of alpha-lactalbumin from the native to a highly ordered compact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entropy of the denaturation of alpha-lactalbumin to compact denatured state are always greater than the enthalpy and entropy of its unfolding. This difference represents the unfolding of the molten globule state. Calorimetric measurements of the heat effect associated with the unfolding of the molten globule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic specificity of the molten globule state, which, in contrast to a protein with unique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and enthalpy of internal interactions. It is concluded that at physiological temperatures the entropy of dehydration is the dominant factor providing stability for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor.     

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.     

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.     

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.     

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.     

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.     

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.     

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.