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O B Ptitsyn - One of the best experts on this subject based on the ideXlab platform.
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Molten Globule like state of cytochrome c under conditions simulating those near the membrane surface
Biochemistry, 1996Co-Authors: V. E. Bychkova, Vladimir N. Uversky, Alexandra E Dujsekina, Stanislav I Klenin, Elisaveta I Tiktopulo, O B PtitsynAbstract:Methanol-induced conformational transitions in cytochrome c have been studied by near- and far-UV circular dichroism, Trp fluorescence, microcalorimetry, and diffusion measurements. The existence of at least two cooperative stages of transition has been shown. At the first stage, the native protein is transformed into an intermediate which has only traces of tertiary structure, but has a native-like secondary structure content and is relatively compact; i.e., it has properties of the Molten Globule state. On the second stage, the alcohol-induced Molten Globule is transformed into a more helical state, typical of proteins at high alcohol concentrations. The conditions at which the alcohol-induced Molten Globule exists (moderately low pH and moderately low dielectric constant) could be similar to those existing near negatively charged membrane surfaces. Consequently, these results might explain how the Molten Globule state can be achieved under physiological conditions.
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further evidence on the equilibrium pre Molten Globule state four state guanidinium chloride induced unfolding of carbonic anhydrase b at low temperature
Journal of Molecular Biology, 1996Co-Authors: Vladimir N. Uversky, O B PtitsynAbstract:Abstract Equilibrium guanidinium chloride-induced unfolding of bovine carbonic anhydrase NB has been investigated by a combination of optical methods with size-exclusion chromatography. It has been shown that, as in the case of staphylococcal β-lactamase, bovine carbonic anhydrase B unfolds at low temperature through two equilibrium intermediates; the Molten Globule and the pre-Molten Globule states. This pre-Molten Globule state has a hydrodynamic volume no more than twofold larger than that of the native state, i.e. is relatively compact. It has a pronounced far UV CD spectrum, suggesting the presence of a substantial secondary structure. It binds 8-anilinonaphthalene-1-sulphonate (though weaker than the Molten Globule state), which suggests the formation of solvent-exposed clusters of non-polar groups. Thus, this novel state of protein molecules shares a number of properties with the “burst” kinetic intermediate of protein folding and can be considered as its equilibrium counterpart.
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further evidence on the equilibrium pre Molten Globule state four state guanidinium chloride induced unfolding of carbonic anhydrase b at low temperature
Journal of Molecular Biology, 1996Co-Authors: Vladimir N. Uversky, O B PtitsynAbstract:Equilibrium guanidinium chloride-induced unfolding of bovine carbonic anhydrase NB has been investigated by a combination of optical methods with size-exclusion chromatography. It has been shown that, as in the case of staphylococcal beta-lactamase, bovine carbonic anhydrase B unfolds at low temperature through two equilibrium intermediates; the Molten Globule and the pre-Molten Globule states. This pre-Molten Globule state has a hydrodynamic volume no more than twofold larger than that of the native state, i.e. is relatively compact. It has a pronounced far UV CD spectrum, suggesting the presence of a substantial secondary structure. It binds 8-anilinonaphthalene-1-sulphonate (though weaker than the Molten Globule state), which suggests the formation of solvent-exposed clusters of non-polar groups. Thus, this novel state of protein molecules shares a number of properties with the "burst" kinetic intermediate of protein folding and can be considered as its equilibrium counterpart.
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Molten Globule and protein folding
Advances in Protein Chemistry, 1995Co-Authors: O B PtitsynAbstract:Publisher Summary This chapter describes the present state of the studies of the Molten Globule state and its role in protein folding and physiological processes. Recent data on the intermediates (equilibrium and kinetic), focusing attention on the Molten Globule state are discussed in the chapter. The Molten Globule has a native-like overall architecture (folding pattern) without including the rigid packing of side chains. It is separated by first-order phase transitions from both the native and the unfolded states and represents a third thermodynamic state of protein molecules. It is a universal kinetic intermediate in protein folding; the structure of this intermediate is similar to the structure of the equilibrium Molten Globule. It is predicted and confirmed experimentally that the Molten Globule state can exist in a living cell and plays an important role in a number of physiological processes.
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retinol binding protein is in the Molten Globule state at low ph
Biochemistry, 1992Co-Authors: V. E. Bychkova, Rodolfo Berni, Gian Luigi Rossi, V P Kutyshenko, O B PtitsynAbstract:Using far- and near-UV circular dichroism, viscosity, tryptophan fluorescence, NMR spectra, binding of a hydrophobic probe, and microcalorimetry, we have shown that the apo form of human retinol-binding protein (RBP) at neutral pH is in a rigid state with properties similar to those of holo-RBP. On the contrary, at acidic pH apo-RBP is in the Molten Globule state which has been earlier revealed for a number of proteins under mild denaturing conditions. We have also shown that, at equilibrium, the pH-induced retinol release from holo-RBP parallels denaturation of the apoprotein. These findings are consistent with our hypothesis that the transformation of RBP into the Molten Globule state is involved in the mechanism whereby retinol is delivered to target cells. In particular, a local acidic pH near the membrane surface of target cells might cause the transition of RBP to the Molten Globule state as well as the release of retinol.
Vladimir N. Uversky - One of the best experts on this subject based on the ideXlab platform.
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The unfolding pathways of the native and Molten Globule states of 5-aminolevulinate synthase.
Biochemical and biophysical research communications, 2016Co-Authors: Bosko M. Stojanovski, Vladimir N. Uversky, Leonid Breydo, Gloria C. FerreiraAbstract:In this communication, we report the equilibrium and kinetic properties of the unfolding pathways of the native (pH 7.5) and alkaline Molten Globule (pH 10.5) states of the pyridoxal 5'-phosphate (PLP)-dependent enzyme 5-aminolevulinate synthase (ALAS). The stability of the Molten Globule state is adversely affected by thermal- and guanidine hydrochloride (GuHCl)-induced denaturation, and the equilibrium unfolding pathways, irrespective of pH, cannot be described with simple two-state models. Rapid kinetic measurements, in the presence of denaturing GuHCl concentrations, reveal that at pH 10.5, the rate of ALAS denaturation is 3 times faster than at pH 7.5. From pH jump experiments, comparable rates for the denaturation of the tertiary structure and PLP-microenvironment were discerned, indicating that the catalytic active site geometry strongly depends on the stable tertiary structural organization. Lastly, we demonstrate that partially folded ALAS tends to self-associate into higher oligomeric species at moderate GuHCl concentrations.
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use of fluorescence decay times of 8 ans protein complexes to study the conformational transitions in proteins which unfold through the Molten Globule state
Biophysical Chemistry, 1996Co-Authors: Vladimir N. Uversky, Stefan Winter, Gunter LoberAbstract:The conformational transitions starting with the native protein, passing the Molten Globule state and finally approaching the unfolded state of proteins was investigated for bovine carbonic anhydrase B (BCAB) and human alpha-lactalbumin (alpha-HLA) by means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential fluorescence decay, where both decay times considerably exceed the decay time of free 8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules attached to the proteins. The fluorescence lifetime of the short-time component is affected by protein association and can be effectively quenched by acrylamide, indicating that 8-ANS molecules preferentially bind at the protein surface. The fluorescence lifetime of the long-time component is independent of the protein and acrylamide concentration and may be related to protein-embedded dye molecules. Changes of the long lifetime component upon GdmCl-induced denaturation and unfolding of BCAB and alpha-HLA correlate well with overall changes of the protein conformation. The transition from native protein to the Molten Globule state is accompanied by an increase of the number of protein-embedded 8-ANS molecules, while the number of dye molecules located at the protein surface decreases. For the transition from the Molten Globule to the unfolded state was the opposite behaviour observed.
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Molten Globule like state of cytochrome c under conditions simulating those near the membrane surface
Biochemistry, 1996Co-Authors: V. E. Bychkova, Vladimir N. Uversky, Alexandra E Dujsekina, Stanislav I Klenin, Elisaveta I Tiktopulo, O B PtitsynAbstract:Methanol-induced conformational transitions in cytochrome c have been studied by near- and far-UV circular dichroism, Trp fluorescence, microcalorimetry, and diffusion measurements. The existence of at least two cooperative stages of transition has been shown. At the first stage, the native protein is transformed into an intermediate which has only traces of tertiary structure, but has a native-like secondary structure content and is relatively compact; i.e., it has properties of the Molten Globule state. On the second stage, the alcohol-induced Molten Globule is transformed into a more helical state, typical of proteins at high alcohol concentrations. The conditions at which the alcohol-induced Molten Globule exists (moderately low pH and moderately low dielectric constant) could be similar to those existing near negatively charged membrane surfaces. Consequently, these results might explain how the Molten Globule state can be achieved under physiological conditions.
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further evidence on the equilibrium pre Molten Globule state four state guanidinium chloride induced unfolding of carbonic anhydrase b at low temperature
Journal of Molecular Biology, 1996Co-Authors: Vladimir N. Uversky, O B PtitsynAbstract:Abstract Equilibrium guanidinium chloride-induced unfolding of bovine carbonic anhydrase NB has been investigated by a combination of optical methods with size-exclusion chromatography. It has been shown that, as in the case of staphylococcal β-lactamase, bovine carbonic anhydrase B unfolds at low temperature through two equilibrium intermediates; the Molten Globule and the pre-Molten Globule states. This pre-Molten Globule state has a hydrodynamic volume no more than twofold larger than that of the native state, i.e. is relatively compact. It has a pronounced far UV CD spectrum, suggesting the presence of a substantial secondary structure. It binds 8-anilinonaphthalene-1-sulphonate (though weaker than the Molten Globule state), which suggests the formation of solvent-exposed clusters of non-polar groups. Thus, this novel state of protein molecules shares a number of properties with the “burst” kinetic intermediate of protein folding and can be considered as its equilibrium counterpart.
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further evidence on the equilibrium pre Molten Globule state four state guanidinium chloride induced unfolding of carbonic anhydrase b at low temperature
Journal of Molecular Biology, 1996Co-Authors: Vladimir N. Uversky, O B PtitsynAbstract:Equilibrium guanidinium chloride-induced unfolding of bovine carbonic anhydrase NB has been investigated by a combination of optical methods with size-exclusion chromatography. It has been shown that, as in the case of staphylococcal beta-lactamase, bovine carbonic anhydrase B unfolds at low temperature through two equilibrium intermediates; the Molten Globule and the pre-Molten Globule states. This pre-Molten Globule state has a hydrodynamic volume no more than twofold larger than that of the native state, i.e. is relatively compact. It has a pronounced far UV CD spectrum, suggesting the presence of a substantial secondary structure. It binds 8-anilinonaphthalene-1-sulphonate (though weaker than the Molten Globule state), which suggests the formation of solvent-exposed clusters of non-polar groups. Thus, this novel state of protein molecules shares a number of properties with the "burst" kinetic intermediate of protein folding and can be considered as its equilibrium counterpart.
Yuji Goto - One of the best experts on this subject based on the ideXlab platform.
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structural characterization of the Molten Globule of alpha lactalbumin by solution x ray scattering
Protein Science, 1997Co-Authors: Mikio Kataoka, Kunihiro Kuwajima, Fumio Tokunaga, Yuji GotoAbstract: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.
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Thermodynamic stability of the Molten Globule states of apomyoglobin.
Journal of Molecular Biology, 1995Co-Authors: Ichiro Nishii, Mikio Kataoka, Yuji GotoAbstract:Abstract Whereas horse apomyoglobin is fully unfolded at pH 2 in the absence of salt, addition of a salt such as sodium chloride or sodium trichloroacetate stabilizes the Molten Globule state. Thermal unfolding of the salt-stabilized Molten Globule states of horse apomyoglobin at pH 2 measured by far-UV circular dichroism occurs not only on heating (i.e. heat-denaturation) but also on cooling (i.e. cold-denaturation). This demonstrates that a hydrophobic interaction contributes to the stability of the Molten Globule state and suggests that the unfolding transition can be represented by a cooperative two-state mechanism. To clarify the mechanism of conformational transition, we investigated the thermal unfolding of the chloride-stabilized Molten Globule state by differential scanning calorimetry. We observed a broad but distinct excess heat capacity peak, which is consistent with the unfolding transition measured by circular dichroism. To further characterize the Molten Globule states, we examined by far-UV circular dichroism the denaturant-induced unfolding transitions of the Molten Globule states stabilized by sodium chloride or sodium trichloroacetate. The urea-induced unfolding transitions of the Molten Globule states were explained by the two-state mechanism. The guanidine-hydrochloride-induced unfolding experiments clarified that the trichloroacetate-stabilized Molten Globule state is distinct from the chloride-stabilized one and that the former involves additional helical segment(s). These results support a view that the thermal unfolding of the Molten Globule states at pH 2 can be approximated by a two-state transition. However, several results suggested that a combined mechanism incorporating the two-state transition and a gradual structural change would be more general in describing the conformational transition of the Molten Globule states. f2
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Structural Characterization of the Molten Globule and Native States of Apomyoglobin by Solution X-ray Scattering
Journal of molecular biology, 1995Co-Authors: Mikio Kataoka, Ichiro Nishii, Tetsuro Fujisawa, Tatzuo Ueki, Fumio Tokunaga, Yuji GotoAbstract:Abstract Compactness and shape are two of the critical properties that describe the degree of protein folding. Solution X-ray scattering is an effective technique for measuring these properties quantitatively. Structural characteristics of various conformational states of horse myoglobin were studied in terms of size and shape by solution X-ray scattering. The radius of gyration for native holomyoglobin was 17.5 A, while that of the apomyoglobin native state was 19.7 A. Corresponding to the increase in the radius of gyration, the largest dimension of the molecule also increased from 47.5 A to 62.5 A. Both states are globular in shape. The scattering profiles in the high angle region suggest that the apomyoglobin native state has a distinct tertiary structure, and that packing of α-helices in the apomyoglobin native state would be looser than that of holomyoglobin. These observations indicate that the native state of apomyoglobin is expanded from that of holomyoglobin, and that the conformations of the two are not identical. The radii of gyration for the acid-unfolded state and the denaturant-unfolded state were 30 A and 35 A, respectively. Both unfolded states have chain-like conformations without any tertiary structures. The radius of gyration and the largest dimension of the Molten Globule stabilized by trichloroacetate were 23.1 A and 72.5 A, respectively. The Molten Globule is expanded from the native state although it is globular, and is much more compact than the unfolded state. The bimodal distance distribution function and scattering profile at high-angle region suggest that the structure of the apomyoglobin Molten Globule contains a core comprising a cluster of multiple α-helices and flaring tail(s), which would be a common structural property of the compact denatured state appearing during the folding process. The compactness of each conformational state is highly correlated with the extent of formation of the α-helix.
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cold denaturation of the Molten Globule states of apomyoglobin and a profile for protein folding
Biochemistry, 1994Co-Authors: Ichiro Nishii, Mikio Kataoka, Fumio Tokunaga, Yuji GotoAbstract:Protein folding is a process in which an extended polypeptide chain acquires compact packing through the formation of specific secondary and tertiary structures and hydrophobic interactions. Although much attention has been paid to secondary and tertiary structures, there is no definitive view about the relationship between these structures, compactness, and hydrophobic interactions during the process of protein folding. We show here that the Molten Globule intermediates of horse apomyoglobin exhibit cold denaturation in addition to heat denaturation, which indicates that the heat capacity change upon unfolding is positive and significant. This demonstrates a small but distinct contribution of hydrophobic interactions to the stability of the Molten Globule state. We determined the radius of gyration of the various conformational states of horse apomyoglobin and holomyoglobin by measuring small angle X-ray scattering. By comparing the conformational states in terms of secondary structure, radius of gyration, and change in heat capacity upon unfolding, we constructed a folding profile. The profile shows that the protein becomes more compact with formation of the secondary structure, but does not form substantial hydrophobic interactions until a later rate-limiting stage when tight packing of the protein side chains occurs. A very similar profile was also obtained with horse cytochrome c. We propose that the folding profile obtained with these proteins will be common to many globular proteins.
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comparison of the conformational stability of the Molten Globule and native states of horse cytochrome c effects of acetylation heat urea and guanidine hydrochloride
Journal of Molecular Biology, 1994Co-Authors: Yoshihisa Hagihara, Yukihiro Tan, Yuji GotoAbstract:The Molten Globule state has been assumed to be a major intermediate of protein folding. We compared the stability of the native and acidic Molten Globule states of horse ferricytochrome c against heat, urea and guanidine hydrochloride (Gdn-HCl) using the intact species and species modified by various degrees of acetylation of the lysyl epsilon-amino groups. After acetylation, the amino groups cannot protonate at acidic pH. Thermal and urea-induced unfolding transitions measured by far-UV circular dichroism and differential scanning calorimetry showed that, whereas acetylation stabilizes the Molten Globule state at pH 2, it destabilizes the native state at pH 7, suggesting a difference in their mechanisms of conformational stability. On the other hand, the effects of Gdn-Hcl were remarkable. Contrary to what was expected from the thermal and urea-induced unfolding transitions, the Gdn-HCl-induced unfolding transition of the native state at pH 7 was insensitive to the extent of acetylation. At pH 2, Gdn-HCl at low concentrations stabilized the Molten Globule state and, at high concentrations, destabilized it. Consideration of the difference in the effects of Gdn-HCl from those of urea or heat indicated that, whereas the net positive charge repulsion destabilizes the Molten Globule state at pH 2, the local negative charge repulsion produced by acetylation of amino groups, and not the net charge, critically destabilizes the native state at pH 7. These results predict that, because of its ionic nature, Gdn-HCl will produce substantially different effects on the conformational states of some proteins compared with those of urea.
Peter S Kim - One of the best experts on this subject based on the ideXlab platform.
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α lactalbumin forms a compact Molten Globule in the absence of disulfide bonds
Nature Structural & Molecular Biology, 1999Co-Authors: Christina Redfield, Peter S Kim, Brenda A Schulman, Michael A Milhollen, Christopher M. DobsonAbstract:Human α-lactalbumin (α-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 α-LA, in which all eight cysteines have been mutated to alanine (all-Ala α-LA), has been studied using NMR spectroscopy. At low pH all-Ala α-LA is nearly as compact as wild type α-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 α-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 α-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.
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a specific hydrophobic core in the alpha lactalbumin Molten Globule
Journal of Molecular Biology, 1998Co-Authors: Peter S KimAbstract:Molten Globules are partially structured protein folding intermediates that adopt a native-like overall backbone topology in the absence of extensive detectable tertiary interactions. It is important to determine the extent of specific tertiary structure present in Molten Globules and to understand the role of specific side-chain packing in stabilizing and specifying Molten-Globule structure. Previous studies indicate that a small degree of specific side-chain packing stabilizes the structures of the cytochrome c, apomyoglobin, and staphylococcal nuclease Molten Globules. Here we investigate the extent of specific side-chain packing in the Molten Globule of α-lactalbumin (α-LA), a highly fluctuating, non-cooperatively formed Molten Globule. By analyzing a set of point mutations in the helical domain of α-LA, we have identified a stabilizing hydrophobic core. Moreover, this core corresponds to a previously identified structural subdomain and likely contains some native-like packing interactions. Our results suggest that native-like packing of core amino acids helps stabilize Molten Globules and that some specific interactions can exist in even highly dynamic, fluctuating species.
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Proline scanning mutagenesis of a Molten Globule reveals non-cooperative formation of a protein's overall topology
Nature Structural Biology, 1996Co-Authors: Brenda A Schulman, Peter S KimAbstract:Small proteins generally fold cooperatively: disruption of significant parts of the folded structure leads to unfolding of the rest of the protein. We show here, using proline scanning mutagenesis, that the native-like tertiary fold of the α-lactalbumin (α-LA) Molten Globule is formed by the non-cooperative assembly of its constituent helices. In contrast to the drastic destabilizing effects of proline substitutions in cooperatively folded proteins, proline mutations in the Molten Globule appear to cause only individual helices to unfold, without significantly influencing the other helices or the overall topology. Thus, the key determinants of a protein's overall fold may not be of the all-or-none type.
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different subdomains are most protected from hydrogen exchange in the Molten Globule and native states of human α lactalbumin
Journal of Molecular Biology, 1995Co-Authors: Brenda A Schulman, Zhengyu Peng, Christina Redfield, Christopher M. Dobson, Peter S KimAbstract:Abstract α-Lactalbumin (α-LA) is a two-domain, calcium-binding protein that forms one of the best studied Molten Globules. We present here amide hydrogen exchange studies of the Molten Globule formed by human α-LA at pH 2 and compare these results with a similar study of the native state at pH|nlbsb|6.3. The most persistent structure in the Molten Globule is localized in the helical domain, consistent ith previous results. However, the helices most protected from hydrogen exchange in the Molten Globule are, in the native state, less protected from exchange than other regions of the protein. The Molten Globule appears to contain major elements of the native fold, but formation of the fully native state requires stabilization of structure around the calcium-binding site and domain interface.
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bipartite structure of the alpha lactalbumin Molten Globule
Nature Structural & Molecular Biology, 1995Co-Authors: Zhengyu Peng, Peter S KimAbstract:Molten Globules are thought to be general intermediates in protein folding. Apparently conflicting studies have failed to clarify whether one of the best characterized Molten Globules, that of alpha-lactalbumin, resembles an expanded native-like protein or a nonspecific collapsed polypeptide. Here we show that the Molten Globule properties of alpha-lactalbumin are largely confined to one of its two domains. The alpha-helical domain forms a helical structure with a native-like tertiary fold, while the beta-sheet domain is largely unstructured. Molten Globules thus possess a native-like backbone topology, but this topology does not necessarily encompass the entire polypeptide chain. Our studies indicate that Molten Globules provide an approximate solution to, and considerable simplification of the protein folding problem.
Kunihiro Kuwajima - One of the best experts on this subject based on the ideXlab platform.
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role of the Molten Globule state in protein folding
Advances in Protein Chemistry, 2000Co-Authors: Munehito Arai, Kunihiro KuwajimaAbstract:Publisher Summary This chapter deals with the structure of the Molten Globules of various globular proteins revealed by the recent experimental studies. Recent advances in experimental techniques, including hydrogen-exchange NMR, solution X-ray scattering, and protein engineering, have provided detailed pictures of the Molten Globules for these proteins. The Molten Globule state has heterogeneous structures, in which one portion of a molecule is more organized and native-like with the other portions being less organized, although the overall structure satisfies the criteria of the Molten Globule state (compactness, the presence of secondary structure, and the lack of rigid tertiary structure). The chapter describes how the Molten Globule state has been identified as the intermediate of kinetic refolding and discusses the kinetic roles of the Molten Globule state in protein folding. The chapter also discusses thermodynamic stability and cooperativity of the Molten Globule state from the viewpoint of the hierarchy of protein folding, in which the Molten Globule state plays a role as a junction of two levels of the hierarchy.
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structural characterization of the Molten Globule of alpha lactalbumin by solution x ray scattering
Protein Science, 1997Co-Authors: Mikio Kataoka, Kunihiro Kuwajima, Fumio Tokunaga, Yuji GotoAbstract: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.
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rapid formation of a Molten Globule intermediate in refolding of α lactalbumin
Folding and Design, 1996Co-Authors: Munehito Arai, Kunihiro KuwajimaAbstract:Backgound: The Molten Globule state is an intermediate between the native and the fully unfolded states of globular proteins and is purported to be an obligatory on-pathway intermediate of protein folding. The Molten Globule state of α -lactalbumin has been best characterized, but two major issues have yet to be clarified. At which stage of the kinetic refolding is the Molten Globule state stably organized? And what is the major driving force that stabilizes the Molten Globule state? We address these questions in this paper. Results We have investigated the refolding kinetics of α -lactalbumin using stopped-flow CD and fluorescence, acrylamide quenching and pulsed hydrogen exchange NMR techniques. A burst-phase intermediate was observed to form within 15 ms. The intermediate was characterized by pronounced, hydrogen-bonded secondary structure, exposure of hydrophobic surfaces and the absence of tertiary structure. Furthermore, the stability of the secondary structure is the same as that in the equilibrium Molten Globule state. Conclusion The burst-phase intermediate in α -lactalbumin refolding is identical with the Molten Globule state. Two different models, the hydrophobic collapse model and the secondary-structure coalescence model, of protein folding are discussed on the basis of the present results. The importance of solvent-separated hydrophobic interactions that stabilize the Molten Globule state is proposed.
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the Molten Globule state of alpha lactalbumin
The FASEB Journal, 1996Co-Authors: Kunihiro KuwajimaAbstract:The Molten Globule state of alpha-lactalbumin is the best-characterized folding intermediate of globular proteins and has been studied intensively by various spectroscopic and physiochemical techniques, including stopped-flow CD and fluorescence spectroscopies, a hydrogen-exchange technique, 1H-NMR spectroscopy, disulfide-exchange chemistry, site-directed mutagenesis, and calorimetric techniques. This review summarizes recent studies. Major findings about the structure of the Molten Globule state are: 1) It is highly heterogeneous, having a highly structured alpha-helical domain with the beta-sheet domain being significantly unfolded; and 2) it is not a nonspecific, collapsed polypeptide but already has a native-like tertiary fold. These structural characteristics are essential to fully understand the thermodynamic properties of the Molten Globule state which are described in connection with a recently proposed computational approach to predict the structure of the Molten Globule state of a protein. Mutant proteins in which the stability of the Molten Globule state was changed were constructed. Studies of the equilibrium unfolding and kinetic refolding of the mutant proteins will provide further insight into the Molten Globule state as a folding intermediate. In spite of an initial expectation that the structure recognized by an Escherichia coli chaperone, GroEL, is the Molten Globule, the interaction of GroEL with alpha-lactalbumin in the Molten Globule state is much weaker than the interaction with more unfolded states of alpha-lactalbumin, a disulfide-reduced form, and disulfide rearranged species.
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the Molten Globule state of alpha lactalbumin
The FASEB Journal, 1996Co-Authors: Kunihiro KuwajimaAbstract:The Molten Globule state of alpha-lactalbumin is the best-characterized folding intermediate of globular proteins and has been studied intensively by various spectroscopic and physiochemical techniques, including stopped-flow CD and fluorescence spectroscopies, a hydrogen-exchange technique, 1H-NMR spectroscopy, disulfide-exchange chemistry, site-directed mutagenesis, and calorimetric techniques. This review summarizes recent studies. Major findings about the structure of the Molten Globule state are: 1) It is highly heterogeneous, having a highly structured alpha-helical domain with the beta-sheet domain being significantly unfolded; and 2) it is not a nonspecific, collapsed polypeptide but already has a native-like tertiary fold. These structural characteristics are essential to fully understand the thermodynamic properties of the Molten Globule state which are described in connection with a recently proposed computational approach to predict the structure of the Molten Globule state of a protein. Mutan...