1H, 13C and 15N resonance assignments for human all-Ala α-lactalbumin in its molten globule and urea-denatured states

Kinetic intermediates that appear early during protein folding often resemble the relatively stable molten globule intermediates formed by several proteins under mildly denaturing conditions. Molten globules have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule that is observed during folding of alpha-beta parallel flavodoxin from Azotobacter vinelandii is a remarkably non-native species. This folding intermediate is helical and contains no beta-sheet and is kinetically off-pathway to the native state. It can be trapped under native-like conditions by substituting residue Phe(44) for Tyr(44). To characterize this species at the residue level, in this study, use is made of interrupted hydrogen/deuterium exchange detected by NMR spectroscopy. In the molten globule of flavodoxin, the helical region comprising residues Leu(110)-Val(125) is shown to be better protected against exchange than the other ordered parts of the folding intermediate. This helical region is better buried than the other helices, causing its context-dependent stabilization against unfolding. Residues Leu(110)-Val(125) thus form the stable core of the helical molten globule of alpha-beta parallel flavodoxin, which is almost entirely structured. Non-native docking of helices in the molten globule of flavodoxin prevents formation of the parallel beta-sheet of native flavodoxin. Hence, to produce native alpha-beta parallel protein molecules, the off-pathway species needs to unfold.

Molten globules

The study of Morjana et al. (1) on a partially unfolded intermediate of protein disulfide isomerase (PDI) raises a number of important points concerning the nature of folding intermediates of globular proteins. PDI is believed to possess two internally homologous domains (2). When subjected to guanidine hydrochloride denaturation, circular dichroism, and fluorescence spectroscopy indicate the presence of a partially folded equilibrium intermediate containing about 40% of the native state's secondary structure. Consistent with this amount ofsecondary structure, these authors suggest that the intermediate may result from unfolding of one of the two structural domains. Based on the fact that the far-UV spectrum is not similar to the native state, these authors argue that this equilibrium intermediate is not a molten globule, a folding intermediate state whose properties have been the subject of much attention and debate (3-5). While one might have expected that all intermediates have similar characteristics, as the PDI study points out, there are many types with rather diverse physical properties; the origins of these differences are not well understood. What differentiates them are the amount of native secondary structure and the degree of side-chain packing specificity. They range from fragments whose properties are essentially identical to the native molecule (6) to those where only a small portion of the molecule has loosely contacting native-like secondary structure (7). In what follows, we summarize what is known about the various types of folding intermediates, discuss what experimental information is required to better characterize them, and describe recent theoretical advances that may provide a fuller understanding of the protein folding process. Perhaps the most expected intermediates are those where a domain or subdomain folds independently and adopts both the native secondary and tertiary structure within this folding unit; i.e., it constitutes a native-like folding module (8-10). PDI may be such a case. However, the best characterized of these native-like modules is the equilibrium folding intermediate seen on urea denaturation of the a-subunit of tryptophan synthase (6, 11) whose native conformation is a single-domain eight-member a/p-barrel (12). This intermediate is located at the N terminus and is composed of six of the eight p-strands plus associated helices. Moreover, the N-terminal fragment folds independently, with properties very similar to the equilibrium intermediate of the entire molecule. Possible insights into the origin of the stability of this intermediate may be obtained from the recent computer simulations of Godzik et al. (13). In these simulations, the local secondary-structure biases are assumed to be consistent with the folded state, but the folding pathway is predicted. These simulations correctly predict the structure of the equilibrium intermediate and indicate that the N-terminal fragment is substantially more stable than the corresponding C-terminal fragment. The equilibrium intermediate basically possesses the secondary structure of the native molecule, but the helices at the fragment interface oscillate and at times cover the exposed hydrophobic core of the molecule. Thus, they act as a cork to impede further assembly. These simulations predict that many, but not all, of the side-chain contacts are identical to those found in the native state. It would be very interesting to see whether this prediction can be verified by experiment. More generally, it appears that the formation of secondary structure found in the native state and the adoption of well-defined patterns of side-chain contacts characteristic of folded proteins are interrelated but separate processes (3-5). Indeed, in the molten-globule state, the molecule is compact, with a volume about 20% larger than native, and has the native-like secondary structure but poorly defined tertiary contacts (10). A key point is whether the entire molecule adopts native-like secondary structure or whether a portion remains unfolded. In both cases, it is unknown whether or not there is a continuous or discrete spectrum of backbone conformations. We first describe the situation when most, if not all, of the native-state secondary structure is present as an intact globule. One example is provided by barnase (14, 15), which prior to the rate-

Recent advances in protein chemistry have led to progress in the understanding of protein folding and properties of possible intermediates during the folding of proteins. The molten globule (MG) state, a major intermediate of protein folding, has a denatured state with native-like secondary structure. In the present work, the acid-induced unfolding of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and its three different variants (G96A, A183T and G96A/A183T) were studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy and 1-anilino naphthalene-8-sulfonate (ANS) binding. At pH<3.0, these EPSPS variants acquire partially folded state, which show the characteristics of the MG state, e.g., a drastic reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding experiments show that hydrophobic surface of these variants is exposed to a greater extent in comparison to the native form, at acidic pH. Wild type, G96A, A183T and G96A/A183T acquire MG states at pH 2.0, 1.5, 3.0 and 3.0, respectively, which show that pH stability of MG state of G96A has increased in comparison to wild type; and pH stability of MG states of two other mutants is lower than that of the wild type. The results suggest that there is a direct relationship between stability of protein and pH stability of its folding intermediates.

Molten globules are compact, partially folded proteins postulated to be general intermediates in protein folding. Human alpha-lactalbumin (alpha-LA) is a two-domain Ca(2+)-binding protein that partially unfolds at low pH to form a molten globule. NMR spectra of molten globules are characterized by broadened resonances due to conformational fluctuations on microsecond to millisecond time scales. These species are often studied at high temperature where NMR resonances are observed to sharpen. The effect of higher temperatures on fast time-scale backbone dynamics of molten globules has not been investigated previously. Here, 1D (15)N direct-detection and 2D indirect-detection (1)H-(15)N heteronuclear NOE experiments have been used to probe fast time-scale dynamics at low and high temperatures for three disulfide-bond variants of human alpha-LA that form molten globules. Disulfide bonds are found to have a significant effect on backbone dynamics within the beta-domain of the molten globule; within the alpha-domain, dynamics are not significantly influenced by these bonds. At 20 degrees C, backbone mobility is significantly decreased in both domains of the molten globule compared to the mobility at 40-50 degrees C. Heteronuclear NOE values determined at 20 degrees C for the alpha-domain are closely similar to those observed for native alpha-LA, indicating that the alpha-LA molten globule has even more native-like character than suggested by studies conducted at higher temperature. Our results highlight the importance of considering the temperature dependence of the molten globule ensemble when making comparisons between experimental data obtained under different conditions.

Elucidation of the mechanism of globular protein folding is a major issue in structural biology, and recent progress in the experimental studies on protein folding has improved understanding of transient structural intermediates along the folding pathway. Recent excellent techniques have shown the presence of the intermediate at an early stage of folding, which has the native-like secondary structure in the same regions as those in the native molecule and the destroyed specific tertiary structure. Also, these intermediates have been shown to be identical with the molten globule (MG) state observed for some proteins such as -lactalbumin ( -LA) as a compact equilibrium unfolding intermediate. Therefore studies on the MG state of -LA are important for understanding protein folding [I]. The native αLA is composed of fourα-helices (A,B,C,and D) and aβ-sheet. Recently it has been shown that in the MG state a native-like backbone topology is partially retained (at least the native Band C -helices), and a nonnative hydrophobic core is formed in a region located between the Band Chelices in the native state. Therefore, to get further information on stabilization of the MG state, the conformational properties of the peptides that encompass the helices and the hydrophobic core region of -LA should be investigated in detail. We synthesized four peptides, B 14(22-33, C 17(84100), C24(84-107), and H7( 101-107), which correspond to the Band C-helices, the C-helix combined with the hydrophobic core, and only the core region, respectively, in bovine LA [2]. Also, to study the folding mechanism of -LA in vivo, interactions of these peptides with a molecular chaperone, GroEL, were studied. The chaperone accelerates in vivo protein folding and inhibits misfolding. It binds to the MG proteins, but the binding mechanism is not yet clear.

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

Volumetric Characterizations of the Native, Molten Globule and Unfolded States of Cytochrome cat Acidic pH

A systematic investigation of the effects of detergents [Sodium dodecyl sulphate (SDS), hexa decyltrimethyl ammonium bromide (CTAB) and Tween-20] on the structure of acid-unfolded papain (EC.3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence, and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits a substantial amount of secondary structure and is relatively less denatured compared with 6 M GdnHCl (guanidine hydrochloride) but loses the persistent tertiary contacts of the native state. Addition of detergents caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Near-UV CD spectra also showed the regain of native-like spectral features in the presence of 8 mM SDS and 3.5 mM CTAB. Induction of structure in acid-unfolded papain was greater in the presence SDS followed by CTAB and Tween-20. Intrinsic tryptophan fluorescence studies indicate the change in the environment of tryptophan residues upon addition of detergents to acid-unfolded papain. Addition of 8 mM SDS resulted in the loss of ANS binding sites exhibited by a decrease in ANS fluorescence intensity, suggesting the burial of hydrophobic patches. Maximum ANS binding was obtained in the presence of 0.1 mM Tween-20 followed by CTAB, indicating a compact "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in the presence of detergents showed the partial recovery of enzymatic activity. These results suggest that papain at low pH and in the presence of SDS exists in a partially folded state characterized by native-like secondary structure and tertiary folds. While in the presence of Tween, acid-unfolded papain exists as a compact intermediate with molten-globule-like characteristics, viz. enhanced hydrophobic surface area and retention of secondary structure. While in the presence of CTAB it exists as a compact intermediate with regain of native-like secondary and partial tertiary structure as well as high ANS binding with the partially recovered enzymatic activity, i.e., a molten globule state with tertiary folds.

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent--2[3H]diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels alpha-lactalbumin as a function of pH (3-7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23-34) and C (86-98) helix of the native structure are extensively labeled. The small beta-domain (40-50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of alpha-lactalbumin, the alpha-domain has a greater degree of persistent structure than the beta-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.

Vibrational Raman Optical Activity of α-Lactalbumin: Comparison with Lysozyme, and Evidence for Native Tertiary Folds in Molten Globule States

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.

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.

Differential scanning calorimetric study of the molten globule state of cytochrome c induced by sodium n-dodecyl sulfate

ABSTRACTThe majority of proteins perform their cellular function after folding into a specific and stable native structure. Additionally, for many proteins less compact ‘molten globule’ states have been observed. Current experimental observations show that the molten globule state can show varying degrees of compactness and solvent accessibility; the underlying molecular cause for this variation is not well understood. While the specificity of protein folding can be studied using protein lattice models, current design procedures for these models tend to generate sequences without molten globule-like behaviour. Here we alter the design process so the distance between the molten globule ensemble and the native structure can be steered; this allows us to design protein sequences with a wide range of folding pathways, and sequences with well-defined heat-induced molten globules. Simulating these sequences we find that (1) molten globule states are compact, but have less specific configurations compared to the folded state, (2) the nature of the molten globule state is highly sequence dependent, (3) both two-state and multi-state folding proteins may show heat-induced molten globule states, as observed in heat capacity curves. The varying nature of the molten globules and typical heat capacity curves associated with the transitions closely resemble experimental observations.