Molten Globule and Protein Folding

Ptitsyn, Oleg B.

doi:10.1016/s0065-3233(08)60546-x

Cited by 1,283 publications

(1,273 citation statements)

References 390 publications

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“…In addition, species 2, 4, 8, 9, and 10 have no proteolytic activity or tertiary structure. All of the characteristics of these forms of ervatamin B are typical of molten globule state (Ptitsyn, 1995), and the states seem to be molten globule-like as in some other proteins (Kuwajima et al, 1976;Nozaka et al, 1978;Kim and Baldwin 1982;Goto et al, 1990a, b;Zerovnik et al, 1999).…”

Section: Discussionmentioning

confidence: 85%

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Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

Sundd¹,

Kundu²,

Jagannadham³

2002

BMB Reports

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The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the α + β class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0-2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a β-sheet conformation and shows a strong binding to 8-anilino-1-napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.

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Section: Discussionmentioning

confidence: 85%

“…However, it has been shown that such phenomenon occurs through some kinetic intermediates that accumulated during the folding process (Ptitsyn, 1995;Gast et al, 1998). Protein folding involves a discrete pathway with the formation of intermediate states between the native and denatured states (Kim and Baldwin, 1990).…”

Section: Introductionmentioning

confidence: 99%

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

Sundd¹,

Kundu²,

Jagannadham³

2002

BMB Reports

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“…Extensive studies of molten globular states of a variety of proteins were initially catalyzed by the realization that elucidating their structural and dynamic features may provide important clues for solving the protein folding problem (2,3). Since the kinetic folding intermediates are populated only transiently during the protein folding process, properties of molten globular states are often studied using the equilibrium models.…”

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confidence: 99%

Structural and Dynamic Characteristics of a Partially Folded State of Ubiquitin Revealed by Hydrogen Exchange Mass Spectrometry

Hoerner

Kaltashov

2005

Biochemistry

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Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Convoluted exchange patterns are interpreted with the aid of HDX simulations in a three-state system (highly structured, partially unstructured, and fully unstructured states). The data clearly indicate a highly dynamic character of the non-native state. Furthermore, combination of HDX and protein ion fragmentation in the gas phase [by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. The highest degree of structural disorder is displayed by the C-terminal segment (Gly 53 -Gly 76 ), which was previously suggested to form a transient R-helix. The least dynamic segment of ubiquitin in the A-state is Thr 9 -Glu 18 (which was previously suggested to form a stable nativelike -strand), with the adjacent segments exhibiting somewhat diminished conformational stability. The study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium.Molten globule is a term that is used to describe a class of compact non-native protein states that are often thought to be general intermediates in protein folding, which are characterized by a substantial degree of secondary structure and a nearly complete absence of a native tertiary fold (1). Extensive studies of molten globular states of a variety of proteins were initially catalyzed by the realization that elucidating their structural and dynamic features may provide important clues for solving the protein folding problem (2,3). Since the kinetic folding intermediates are populated only transiently during the protein folding process, properties of molten globular states are often studied using the equilibrium models. In fact, it is the partially unfolded equilibrium intermediates that were initially proposed to belong to a common physical state of globular proteins, for which the term molten globular state was coined. More recently, renewed interest in the properties and behavior of molten globular states was instigated by a growing understanding that structural disorder is quite ubiquitous in vivo and often plays an important role in a variety of physiological processes (4). Such partially unstructured protein states have been implicated in a variety of processes ranging from protein translocation through membranes to ordered oligomerization, recognition, signaling, and even catalysis.The experimental tools employed in the studies of molten globular states include circular dichroism spe...

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“…Indeed, evidence has been provided that TFE, and other alcohols, can induce the a-helical conformation in globular proteins consisting mainly of @sheets (Dufour & Haertlk, 1990;Fan et al, 1993;Liu et al, 1994;Shiraki et al, 1994). Overall, these various conformational properties of the TFE state can be related to those of the "molten globule" state of a protein, in which a polypeptide chain acquires a high content of secondary structure, and the side chains do not have fixed conformations (Kuwajima, 1989;Ptitsyn, 1992Ptitsyn, , 1995.…”

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confidence: 99%

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42"C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Thl) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Thl maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-ATm 16 "C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Thl is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Thl retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 3 1-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a &strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Thl and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al., 1975;

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Molten Globule and Protein Folding

Cited by 1,283 publications

References 390 publications

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

Structural and Dynamic Characteristics of a Partially Folded State of Ubiquitin Revealed by Hydrogen Exchange Mass Spectrometry

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

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