Compactness of the kinetic molten globule of bovineα‐lactalbumin: A dynamic light scattering study

Gast, Klaus; Zirwer, Dietrich; Müller-Frohne, Marlies; Damaschun, G.

doi:10.1002/pro.5560070917

Cited by 59 publications

(59 citation statements)

References 35 publications

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“…For polydisperse samples, each particle size contributes its own exponential decay to the field correlation function of the scattered light. This function can be expressed as [24] where G( ) d is the intensity scattered by the particles with a decay constant between and + d . Deriving the desired information from Eq.…”

Section: Dynamic Light Scatteringmentioning

confidence: 99%

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The Effect of Solute Concentration on Equilibrium Partitioning in Polymeric Gels

Gerhardt

Dungan

Phillips

2001

Journal of Colloid and Interface Science

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Section: Dynamic Light Scatteringmentioning

confidence: 99%

“…Deriving the desired information from Eq. [24] (i.e., the distribution of decay G( ) of decay constant ) requires an inversion of the Laplace transform. Laplace inversion of the correlation curves was performed using a constrained regularization program (REPES) to obtain the distribution of decay times.…”

Section: Dynamic Light Scatteringmentioning

confidence: 99%

The Effect of Solute Concentration on Equilibrium Partitioning in Polymeric Gels

Gerhardt

Dungan

Phillips

2001

Journal of Colloid and Interface Science

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“…Like most denatured or partially denatured state of proteins (30,31), it comprises a large number of conformational isomers. So far, characterization of the structure of denatured ␣-LA and the molten globule state of ␣-LA have been achieved by measuring the average property of these collective isomers using a wide range of spectroscopic and physiochemical methods, including circular dichroism (1,2,19,32), fluorescence (12,32,33), NMR (5,14,15,20,29,34), disulfide replacement (35)(36)(37), limited proteolysis (38,39), light scattering (40,41), and calorimetric techniques (42). Further understanding of the denatured state of ␣-LA and the molten globule state of ␣-LA will require fractionation of diverse populations of conformational isomers that constitute the denatured ␣-LA.…”

Section: ␣-Lamentioning

confidence: 99%

The Structure of Denatured α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

Chang

2001

Journal of Biological Chemistry

100

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The structure of denatured ␣-lactalbumin (␣-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, ␣-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured ␣-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured ␣-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of ␣-LA. Most importantly, unique isomers of partially unfolded ␣-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic ␣-helical domain, generating a predominant isomer containing two native disulfide bonds at the ␤-sheet domain and two scrambled disulfide bonds at the ␣-helical region. Thermal denaturation selectively unfolds the ␤-sheet domain of ␣-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of ␣-LA. ␣-LA1 represents one of the most extensively investigated models for understanding the mechanism of protein stability, folding, and unfolding. Under a variety of conditions, ␣-LA adopts a partially structured conformation termed as "molten globule" (1-9), which has been observed along both the unfolding (1, 2, 5, 10 -12) and folding pathways (13-18) of ␣-LA and is considered one of the best characterized folding/unfolding intermediates of globular proteins. At low pH (1,19,20), elevated temperature (19, 21), or mild concentration of denaturant (e.g. GdmCl; Refs. 22, 23), the native ␣-LA unfolds to the state of a molten globule. During the refolding, the fully denatured ␣-LA undergoes a rapid collapse to the state of molten globule (13,14,18) as intermediate, which is followed by consolidation and search of side chain interactions to attain the native protein (14). The structure of ␣-LA molten globule is characterized by a high degree of native-like secondary structure and a fluctuated tertiary fold (1)(2)(3)(4)19). It is stabilized mainly by the core hydrophobic amino acids within the ␣-helical domain (24 -28) and may form even in the absence of its four disulfide bonds (15). The structure of the molten globule of ␣-LA is also highly heterogeneous (3, 27, 29). Like most denatured or partially denatured state of proteins (30, 31), it comprises a large number of conformational isomers. So far, characterization of the structure of denatured ␣-LA and the molten globule state of ␣-LA have been achieved by measuring the average property of these collective isomers using a wide range of spectroscopic and physiochemical methods, including circular dichroism (1, 2, ...

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“…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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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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Compactness of the kinetic molten globule of bovineα‐lactalbumin: A dynamic light scattering study

Cited by 59 publications

References 35 publications

The Effect of Solute Concentration on Equilibrium Partitioning in Polymeric Gels

The Effect of Solute Concentration on Equilibrium Partitioning in Polymeric Gels

The Structure of Denatured α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

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

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