Macromolecular crowding-induced molten globule states of the alkali pH-denatured proteins

Kumar, Rajesh; Sharma, Deepak; Garg, Mansi; Kumar, Vinay; Agarwal, Mukesh Chand

doi:10.1016/j.bbapap.2018.08.012

Cited by 9 publications

(5 citation statements)

References 119 publications

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“…Numerous studies using macromolecules such as Ficoll, dextran, and poly(ethylene glycols) as crowders, mimicking cellular crowding, are providing insights into the role of both entropic and enthalpic interactions in modulating the structure, stability, function, and conformational dynamics of the proteins. − The interactions due to crowding may be driven by various interatomic interactions, including charge–charge, hydrophobic, and polar contacts, which can favor either the folded or the unfolded states, leading to a perturbation in their structure, stability, binding, dynamics, and function. ,− Currently, contrasting observations imply the role of both volume exclusion (entropic) and soft interactions (enthalpic) in modulating the structure, stability, and function, leading to new perspectives and questions. − The effect of crowders depends on the nature, size of the crowder, and the protein itself. , Further, crowding not only can modulate conformational stability but can also affect the equilibria between the accessible conformational states. , Along similar lines, recent data indicate that crowding can induce stable non-native states. − …”

Section: Introductionmentioning

confidence: 99%

Crowding by Poly(ethylene glycol) Destabilizes Chemotaxis Protein Y (CheY)

2022

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The prevailing understanding of various aspects of biochemical processes, including folding, stability, intermolecular interactions, and the binding of metals, substrates, and inhibitors, is derived from studies carried out under dilute and homogeneous conditions devoid of a crowding-related environment. The effect of crowding-induced modulation on the structure and stability of native and magnesium-dependent Chemotaxis Y (CheY), a bacterial signaling protein, was probed in the presence and absence of poly(ethylene glycol) (PEG). A combined analysis from circular dichroism, intrinsic and extrinsic fluorescence, and tryptophan fluorescence lifetime changes indicates that PEG perturbs the structure but leaves the thermal stability largely unchanged. Intriguingly, while the stability of the protein is enhanced in the presence of magnesium under dilute buffer conditions, PEG-induced crowding leads to reduced thermal stability in the presence of magnesium. Nuclear magnetic resonance (NMR) chemical shift perturbations and resonance broadening for a subset of residues indicate that PEG interacts specifically with a subset of hydrophilic and hydrophobic residues found predominantly in α helices, β strands, and in the vicinity of the metal-binding region. Thus, PEG prompted conformational perturbation, presumably provides a different situation for magnesium interaction, thereby perturbing the magnesium-prompted stability. In summary, our results highlight the dominance of enthalpic contributions between PEG and CheY via both hydrophilic and hydrophobic interactions, which can subtly affect the conformation, modulating the metal–protein interaction and stability, implying that in the context of cellular situation, structure, stability, and magnesium binding thermodynamics of CheY may be different from those measured in dilute solution.

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Section: Introductionmentioning

confidence: 99%

Crowding by Poly(ethylene glycol) Destabilizes Chemotaxis Protein Y (CheY)

2022

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“…Glycerol addition increased the volumetric pod activity by enhancing the cell density, but pod activity was still decreased at pH 6.0, possibly owing to the effects of macromolecular crowding. 33 The proportion of soluble pod in cells did not differ at different glycerol concentrations (Fig. 7c).…”

Section: Discussionmentioning

confidence: 84%

“…Herein, total pod activity was increased to 21 243.3 U L −1 by maintaining the pH at 6.0 and adding glycerol to increase the cell density. Glycerol addition increased the volumetric pod activity by enhancing the cell density, but pod activity was still decreased at pH 6.0, possibly owing to the effects of macromolecular crowding . The proportion of soluble pod in cells did not differ at different glycerol concentrations (Fig.…”

Section: Discussionmentioning

confidence: 93%

Soluble recombinant pyruvate oxidase production in Escherichia coli can be enhanced and inclusion bodies minimized by avoiding pH stress

Zhang

2019

J of Chemical Tech & Biotech

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BACKGROUND: Recombinant pyruvate oxidase (pod) is in high demand owing to its broad application in biochemical analysis and low yield from wild microbial strains. Recombinant Escherichia coli harboring pET28a-pod is an efficient producer of pod, facilitating its production in high yield. Optimizing cultivation using shake flasks is necessary for high-level soluble pod production. RESULTS:During recombinant E. coli cultivation, 96.9% of pod activity was lost, and 3.1% of residual pod activity remained in a 25 mL working volume in 250 mL flasks owing to accumulation of pod in insoluble inclusion bodies. pH stress was the factor responsible for this phenomenon based on investigating dissolved oxygen and carbon source addition. Residual pod activity was increased to 43.3, 88.0 and 61.5% by maintaining the pH at 5.0, 6.0 and 7.0 respectively during the induction phase, which decreased inclusion body formation and increased production of soluble pod. Glycerol addition increased the cell density and volumetric pod activity to 21 243.3 U L −1 at pH 6.0 after 64 h induction. CONCLUSION: Soluble pod is converted to inclusion bodies under pH stress conditions, and this can be avoided by pH maintenance.

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“…However, fluorescence studies revealed that this is actually an MG. Perhaps, similarly, formation of this MG under the crowded conditions of the cell "may increase the rate of the transition towards the DNA bound state and facilitate H1 diffusion inside cell nuclei" [272]. Alkali pH-unfolded ferricytochrome c and lysozyme at pH 12.9 (±0.1) were shown to adopt MG conformations in the presence of various crowding agents (Dextran-40, Dextran-70, and Ficoll-70) [273].…”

Section: Macromolecular Crowding and Molten Globulesmentioning

confidence: 99%

Pre-Molten, Wet, and Dry Molten Globules en Route to the Functional State of Proteins

Gupta

Uversky

2023

IJMS

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Transitions between the unfolded and native states of the ordered globular proteins are accompanied by the accumulation of several intermediates, such as pre-molten globules, wet molten globules, and dry molten globules. Structurally equivalent conformations can serve as native functional states of intrinsically disordered proteins. This overview captures the characteristics and importance of these molten globules in both structured and intrinsically disordered proteins. It also discusses examples of engineered molten globules. The formation of these intermediates under conditions of macromolecular crowding and their interactions with nanomaterials are also reviewed.

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Macromolecular crowding-induced molten globule states of the alkali pH-denatured proteins

Cited by 9 publications

References 119 publications

Crowding by Poly(ethylene glycol) Destabilizes Chemotaxis Protein Y (CheY)

Crowding by Poly(ethylene glycol) Destabilizes Chemotaxis Protein Y (CheY)

Soluble recombinant pyruvate oxidase production in Escherichia coli can be enhanced and inclusion bodies minimized by avoiding pH stress

Pre-Molten, Wet, and Dry Molten Globules en Route to the Functional State of Proteins

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