The Burst-phase Intermediate in the Refolding of β-Lactoglobulin Studied by Stopped-flow Circular Dichroism and Absorption Spectroscopy

Kuwajima, Kunihiro; Yamaya, Hidetoshi; Sugai, Shintaro

doi:10.1006/jmbi.1996.0678

Cited by 126 publications

(119 citation statements)

References 84 publications

(163 reference statements)

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“…The native L-PGDS was composed of 17% ␣-helix, 45% ␤-sheet, and 38% coil. The secondary structure contents of L-PGDS are similar to those of ␤-lactoglobulin, a member of the lipocalin family, estimated from both CD spectra and x-ray crystallography (30). In the presence of 0.5 M GdnHCl or 1 M urea, the ␤-sheet content increased from 45 to 51 or 50%, respectively, whereas the coil contents decreased from 38 to 32 or 32%, respectively, without any changes in the ␣-helical structures.…”

Section: Two Equilibrium States Of L-pgds In the Unfoldingmentioning

confidence: 59%

“…␤-Lactoglobulin, a member of the lipocalin family, also exhibits a broad selectivity of ligand binding (41), but it does not bind bilirubin or biliverdin (data not shown). In previous unfolding studies on ␤-lactoglobulin by CD analysis (30,42), the ellipticities at 219 and 222 nm were shown to be increased in the presence of GdnHCl up to 2.5 M, as compared with the value in the absence of GdnHCl. Although the increase in the secondary structure of ␤-lactoglobulin was not examined in those studies, such results indicate that the ␤-sheet content in ␤-lactoglobulin was increased by low concentrations of GdnHCl and suggest that the activity enhanced-like equilibrium state was also formed during the unfolding of ␤-lactoglobulin.…”

Section: Two Equilibrium States Of L-pgds In the Reversible Foldingmentioning

confidence: 85%

See 1 more Smart Citation

Characterization of the Unfolding Process of Lipocalin-type Prostaglandin D Synthase

Inui¹,

Ohkubo²,

Emi³

et al. 2003

Journal of Biological Chemistry

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Section: Two Equilibrium States Of L-pgds In the Unfoldingmentioning

confidence: 59%

Section: Two Equilibrium States Of L-pgds In the Reversible Foldingmentioning

confidence: 85%

Characterization of the Unfolding Process of Lipocalin-type Prostaglandin D Synthase

Inui¹,

Ohkubo²,

Emi³

et al. 2003

Journal of Biological Chemistry

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“…There have been few reports of non-native intermediates in proteins (Liu et al, 1994;Logan et al, 1994;Hamada et al, 1996;Kuwajima et al, 1996;Mendieta et al, 1999). The solvent composition must be a very important factor in determining the secondary structure of a given amino acid sequence in vitro.…”

Section: Thermal Unfolding By Spectroscopymentioning

confidence: 99%

Alcohol and Temperature Induced Conformational Transitions in Ervatamin B: Sequential Unfolding of Domains

Kundu¹,

Sundd²,

Jagannadham³

2002

BMB Reports

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The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly α-helical to β-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the α-helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.

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“…The pathway model emphasizes narrow pathways in the conformational space, whereas the funnel theory focuses on quick folding on a funnel-like potential surface. A number of experimental results, particularly for small proteins, have been interpreted in terms of either the pathway or funnel model (4)(5)(6)(7)(8)(9)(10)(11). However, these views do not necessarily present a comprehensive picture explaining in detail how a specific protein folds into the native structure.…”

mentioning

confidence: 99%

Phylogeny of protein-folding trajectories reveals a unique pathway to native structure

Ota

Ikeguchi²,

Kidera³

2004

Proc. Natl. Acad. Sci. U.S.A.

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To scrutinize how a protein folds at atomic resolution, we performed 200 molecular dynamics simulations (each of 50 ns) of the miniprotein Trp-cage on the computational grid. Within the trajectories, 58 folding and 31 unfolding events were identified and subjected to extensive comparison and classification. Based on an analogy with biological sequences, the folding and unfolding trajectories (arrays of sequential snapshots of structures) were aligned by dynamic programming allowing gaps. A phylogenetic tree derived from the alignments revealed four distinct groups of the trajectories, characterized by the Trp side-chain motions and the main-chain motions. It was found that only one group attained the native structure and that the other three led to pseudonative structures having the correct main-chain trace but different nonnative Trp side-chain rotamers, indicating that those four folded structures were each attained through a unique folding pathway.grid computing ͉ simulation ͉ trajectory alignment ͉ Trp-cage P rotein molecules rapidly fold into a unique, native structure despite the vast size of conformational space in the unfolded state (1). To resolve the paradox between the large space and the fast process, the folding pathway (2) and folding funnel (3) models have been proposed. The pathway model emphasizes narrow pathways in the conformational space, whereas the funnel theory focuses on quick folding on a funnel-like potential surface. A number of experimental results, particularly for small proteins, have been interpreted in terms of either the pathway or funnel model (4-11). However, these views do not necessarily present a comprehensive picture explaining in detail how a specific protein folds into the native structure. Recently, folding simulation at atomic resolution has become a realistic possibility to study the folding process of small proteins (12-18) in spite of the extremely large computational burden. An atomically detailed picture of the folding process could explain how a specific protein folds and complement the generic theories.The difficulty in simulating folding lies not only in the relatively short simulated time scale but also in two other issues. The first is the representation of the highly stochastic nature of protein motion. A single observation of the folding event cannot be a representative of various trajectories in the folding process. An ensemble comprising a diversity of folding trajectories is indispensable to develop a rigorous understanding. The second problem is how to analyze the complicated folding trajectories in a high-dimensional space. Projection of the motions onto two-to three-dimensional space has been used for small peptides (19,20), but it does not have high enough resolution to depict detailed dynamical features in a larger system. We need a more sophisticated technique to analyze large sets of trajectory data.In this study we calculated an ensemble of folding trajectories of a small protein, a 20-residue miniprotein Trp-cage [TC5b; NLYIQ WLKDG GPSSG RPPPS; P...

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The Burst-phase Intermediate in the Refolding of β-Lactoglobulin Studied by Stopped-flow Circular Dichroism and Absorption Spectroscopy

Cited by 126 publications

References 84 publications

Characterization of the Unfolding Process of Lipocalin-type Prostaglandin D Synthase

Characterization of the Unfolding Process of Lipocalin-type Prostaglandin D Synthase

Alcohol and Temperature Induced Conformational Transitions in Ervatamin B: Sequential Unfolding of Domains

Phylogeny of protein-folding trajectories reveals a unique pathway to native structure

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