Polyol-induced molten globule of cytochrome c: an evidence for stabilization by hydrophobic interaction

Kamiyama, Tadashi; Sadahide, Yosuke; Nogusa, Yoshihito; Gekko, Kunihiko

doi:10.1016/s0167-4838(99)00159-4

Cited by 106 publications

(68 citation statements)

References 46 publications

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“…However, its effect on CS refolding is almost similar to that of sorbitol. Like sorbitol, xylitol has also been shown to stabilize the molten globule state of cytochrome c (58). A higher concentration of xylitol (2 M) led to an appreciable decrease in the refolding yield of CS, which again points to factors like the solvent viscosity being responsible for affecting the subunit folding kinetics and in turn preventing proteinprotein association.…”

Section: Effect Of Polyols On the Cs Refolding Yield-the Data Inmentioning

confidence: 99%

“…Increases in the T m of globular proteins by sorbitol are due to their preferential hydration, which is a consequence of the increase in the surface tension of solvent water (41). Polyols, including sorbitol, have been shown to stabilize the acid-unfolded molten globule state of cytochrome c by strengthening hydrophobic interactions that overcome electrostatic repulsion at low pH between charged residues (58). In a recent study on the nucleocapsid protein of rhabdovirus, sorbitol has been shown to reduce aggregation after expression of the protein in Escherichia coli.…”

Section: Effect Of Polyols On the Cs Refolding Yield-the Data Inmentioning

confidence: 99%

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Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes

Mishra

Seckler

Bhat

2005

Journal of Biological Chemistry

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Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 M glycerol and 10 g/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 M. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.Aggregation of proteins during folding, both in vitro and in vivo, is known to lead to low native protein yields as well as the onset of several age-related diseases (1). Hence, there is a growing interest in developing strategies to prevent protein aggregation to enhance protein refolding yields and to design drugs for diseases involving protein aggregation. Several attempts have been made in this direction with successes as well as failures (2-4). In this study, we used citrate synthase (CS), 1 a non-disulfide-bonded dimeric protein (ϳ100 kDa) highly prone to aggregation during in vitro refolding (5-12), as a model protein. The aggregation-prone nature of CS during its refolding has made it an attractive model system to study the effect of molecular chaperones on the prevention of its aggregation and to develop strategies for enhancing protein refolding yields (8,(13)(14)(15)(16)(17)(18)(19)(20). Using the molecular chaperones GroEL and GroES, up to 80% refolding of CS could be obtained at 10 g/ml protein, whereas unassisted refolding was only 5% (7). Inspired by the GroEL/ES-assisted two-step folding mechanism, Rozema and Gellman (9) proposed an artificial chaperone-assisted ref...

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Section: Effect Of Polyols On the Cs Refolding Yield-the Data Inmentioning

confidence: 99%

Section: Effect Of Polyols On the Cs Refolding Yield-the Data Inmentioning

confidence: 99%

Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes

Mishra

Seckler

Bhat

2005

Journal of Biological Chemistry

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“…Bolen and Baskakov demonstrated that the addition of certain osmolytes results in the compaction of RNase A [60]. Polyols have been shown to stabilize the acidunfolded molten globule state of cytochrome c by enhancing hydrophobic interactions that overcome electrostatic repulsion at low pH between charged residues [61].…”

Section: Polyolsmentioning

confidence: 99%

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

Alibolandi¹,

Mirzahosei²

2011

American J. of Biochemistry and Molecular Biology

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Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

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“…Cyt c, like other acid denatured proteins is maximally unfolded at pH 2.0 by HCl in the absence of salts and undergoes a cooperative transformation to an intermediate structure called the A-state [18] which has properties typical of the molten globule [5,19] upon addition of anions from either salts or acids. The molten globule state or the A-state of cyt c has been studied extensively in recent years by a variety of techniques which has enabled its main features to be defined [15,16,[20][21][22][23]. The A-state of cyt c has been reported in acidic solution containing high salt concentration [2,14,24] or through neutralization of charges by acetylation [25,26].…”

Section: Introductionmentioning

confidence: 99%