Low crowding agent concentration destabilizes against pressure unfolding

Somkuti, Judit; Török, Zsófia; Pfalzgraf, F.; Smeller, László

doi:10.1016/j.bpc.2017.04.013

Cited by 8 publications

(7 citation statements)

References 45 publications

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“…The addition of inert macromolecules into the culture media, by emulating the naturally crowded in vivo milieu, amplifies deposition of cellsecreted ECM. [412] Polydis persed macromolecules have been shown to be more effective with respect to ECM deposition, due to more efficient volume exclusion effect. [413] To date, macromolecular crowding has been shown to enhance ECM deposition in permanently differ entiated cell culture [414] and in naïve stem cell culture [415] and to enhance adipogenesis in adiposeinduced stem cell culture.…”

Section: Tissue Engineered Structuresmentioning

confidence: 99%

“…In the dilute culture media, this procollagen/collagen conversion is very slow. The addition of inert macromolecules into the culture media, by emulating the naturally crowded in vivo milieu, amplifies deposition of cell‐secreted ECM . Polydispersed macromolecules have been shown to be more effective with respect to ECM deposition, due to more efficient volume exclusion effect .…”

Section: Collagen Scaffoldsmentioning

confidence: 99%

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The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development

et al. 2018

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Collagen is the oldest and most abundant extracellular matrix protein that has found many applications in food, cosmetic, pharmaceutical, and biomedical industries. First, an overview of the family of collagens and their respective structures, conformation, and biosynthesis is provided. The advances and shortfalls of various collagen preparations (e.g., mammalian/marine extracted collagen, cell‐produced collagens, recombinant collagens, and collagen‐like peptides) and crosslinking technologies (e.g., chemical, physical, and biological) are then critically discussed. Subsequently, an array of structural, thermal, mechanical, biochemical, and biological assays is examined, which are developed to analyze and characterize collagenous structures. Lastly, a comprehensive review is provided on how advances in engineering, chemistry, and biology have enabled the development of bioactive, 3D structures (e.g., tissue grafts, biomaterials, cell‐assembled tissue equivalents) that closely imitate native supramolecular assemblies and have the capacity to deliver in a localized and sustained manner viable cell populations and/or bioactive/therapeutic molecules. Clearly, collagens have a long history in both evolution and biotechnology and continue to offer both challenges and exciting opportunities in regenerative medicine as nature's biomaterial of choice.

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Section: Tissue Engineered Structuresmentioning

confidence: 99%

Section: Collagen Scaffoldsmentioning

confidence: 99%

The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development

et al. 2018

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“…Small molecules lack significant excluded volume; oversize crowders have large interstitial spaces in which proteins are not crowded. At low concentration, crowders in vitro can actually destabilize proteins during temperature or pressure denaturation …”

Section: Stresses That Affect Folding and Binding In Vitromentioning

confidence: 99%

“…At low concentration, crowders in vitro can actually destabilize proteins during temperature or pressure denaturation. 68 In vitro crowding by substances such as Ficoll, a cross-linked carbohydrate, can induce structural transitions in proteins, affect enzyme catalysis, and lead to the formation of entirely new structural ensembles in the protein's P−T−crowding phase diagram. 37…”

Section: Stresses That Affect Folding and Binding In Vitromentioning

confidence: 99%

Proteins: “Boil ’Em, Mash ’Em, Stick ’Em in a Stew”

2019

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Cells of the vast majority of organisms are subject to temperature, pressure, pH, ionic strength, and other stresses. We discuss these effects in the light of protein folding and protein interactions in vitro, in complex environments, in cells, and in vivo. Protein phase diagrams provide a way of organizing different structural ensembles that occur under stress and how one can move among ensembles. Experiments that perturb biomolecules in vitro or in cells by stressing them have revealed much about the underlying forces that are competing to control protein stability, folding, and function. Two phenomena that emerge and serve to broadly classify effects of the cellular environment are crowding (mainly due to repulsive forces) and sticking (mainly due to attractive forces). The interior of cells is closely balanced between these emergent effects, and stress can tip the balance one way or the other. The free energy scale involved is small but significant on the scale of the “on/off switches” that control signaling in cells or of protein–protein association with a favorable function such as increased enzyme processivity. Quantitative tools from biophysical chemistry will play an important role in elucidating the world of crowding and sticking under stress.

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“…It is commonly hypothesized that such a crowded environment promotes compact protein conformations, thus favoring substrate metabolism . Previous studies on macromolecular crowding effects have mostly focused on protein folding, structure, and stability of soluble proteins . For instance, it was shown that macromolecular crowding, could affect subunit association of the tetrameric protein GAPD .…”

Section: Introductionmentioning

confidence: 99%

Lipid composition and macromolecular crowding effects on CYP2J2‐mediated drug metabolism in nanodiscs

2019

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Lipid composition and macromolecular crowding are key external effectors of protein activity and stability whose role varies between different proteins. Therefore, it is imperative to study their effects on individual protein function. CYP2J2 is a membrane-bound cytochrome P450 in the heart involved in the metabolism of fatty acids and xenobiotics. In order to facilitate this metabolism, cytochrome P450 reductase (CPR), transfers electrons to CYP2J2 from NADPH. Herein, we use nanodiscs to show that lipid composition of the membrane bilayer affects substrate metabolism of the CYP2J2-CPR nanodisc (ND) system. Differential effects on both NADPH oxidation and substrate metabolism by CYP2J2-CPR are dependent on the lipid composition. For instance, sphingomyelin containing nanodiscs produced more secondary substrate metabolites than discs of other lipid compositions, implying a possible conformational change leading to processive metabolism. Furthermore, we demonstrate that macromolecular crowding plays a role in the lipid-solubilized CYP2J2-CPR system by increasing the K m and decreasing the V max , and effect that is size-dependent. Crowding also affects the CYP2J2-CPR-ND system by decreasing both the K m and V max for Dextran-based macromolecular crowding agents, implying an increase in substrate affinity but a lack of metabolism. Finally, protein denaturation studies show that crowding agents destabilize CYP2J2, while the multidomain protein CPR is stabilized. Overall, these studies are the first report on the role of the surrounding lipid environment and macromolecular crowding in modulating enzymatic function of CYP2J2-CPR membrane protein system. Additional Supporting Information may be found in the online version of this article. Statement of importance:This work is of interest to researchers studying macromolecular crowding, protein-lipid interactions, and cytochrome P450s. This work provides key insights into the kinetic effects of lipid composition as well as macromolecular crowding, a constant force within any cell. We also determine the effects of a crowded environment on membrane protein stability. As CYPs are important drug metabolizers, such data are pertinent to current and future studies.

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Low crowding agent concentration destabilizes against pressure unfolding

Cited by 8 publications

References 45 publications

The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development

The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development

Proteins: “Boil ’Em, Mash ’Em, Stick ’Em in a Stew”

Lipid composition and macromolecular crowding effects on CYP2J2‐mediated drug metabolism in nanodiscs

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