Residue level quantification of protein stability in living cells

Monteith, William B.; Pielak, Gary J.

doi:10.1073/pnas.1406845111

Cited by 106 publications

(187 citation statements)

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“…These studies have, in particular, shown that crowding can cause an either upward or downward shift in the stability of folded test proteins, [7][8][9][11][12][13][14][15][16] and the direction of the shift can, in part, be rationalized based on net-charge considerations. 9 The protein of the present study, trp-cage, has a well-defined folded structure but is small and malleable. In the previous work by Predeus et al, 44 this protein was studied in the presence of protein GB1 crowders by molecular dynamics simulations, at a temperature where it is folded under dilute conditions.…”

Section: Discussionmentioning

confidence: 99%

“…[1][2][3][4][5] It is well established that excluded-volume interactions with crowder particles can have a significant stabilizing effect on globular proteins, as demonstrated by experiments with synthetic crowding agents such as Ficoll or dextran. 6 Today, experiments are increasingly often performed under more realistic crowding conditions, using concentrated protein solutions [7][8][9][10] or cells. 9,[11][12][13][14][15][16] In these systems, it has been shown that other interactions may dominate over the steric repulsions and lead to a net destabilization of globular proteins.…”

Section: Introductionmentioning

confidence: 99%

“…6 Today, experiments are increasingly often performed under more realistic crowding conditions, using concentrated protein solutions [7][8][9][10] or cells. 9,[11][12][13][14][15][16] In these systems, it has been shown that other interactions may dominate over the steric repulsions and lead to a net destabilization of globular proteins. [7][8][9][12][13][14]16 For a fundamental understanding of these effects, there is a need for computational approaches that permit the simulation of folding/unfolding equilibria in the presence of complex biomolecular crowding agents.…”

Section: Introductionmentioning

confidence: 99%

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Equilibrium simulation of trp-cage in the presence of protein crowders

Bille

Linse

Mohanty

et al. 2015

The Journal of Chemical Physics

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Link to publication Citation for published version (APA): Bille, A., Linse, B., Mohanty, S., & Irbäck, A. (2015). Equilibrium simulation of trp-cage in the presence of protein crowders. Journal of Chemical Physics, 143(17), [175102]. DOI: 10.1063/1.4934997General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. While steric crowders tend to stabilize globular proteins, it has been found that protein crowders can have an either stabilizing or destabilizing effect, where a destabilization may arise from nonspecific attractive interactions between the test protein and the crowders. Here, we use Monte Carlo replicaexchange methods to explore the equilibrium behavior of the miniprotein trp-cage in the presence of protein crowders. Our results suggest that the surrounding crowders prevent trp-cage from adopting its global native fold, while giving rise to a stabilization of its main secondary-structure element, an α-helix. With the crowding agent used (bovine pancreatic trypsin inhibitor), the trp-cage-crowder interactions are found to be specific, involving a few key residues, most of which are prolines. The effects of these crowders are contrasted with those of hard-sphere crowders. C 2015 AIP Publishing LLC. Equilibrium simulation of trp-cage in the presence of protein crowders[http://dx

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Equilibrium simulation of trp-cage in the presence of protein crowders

Bille

Linse

Mohanty

et al. 2015

The Journal of Chemical Physics

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“…Compared with the ideal (dilute) environments conventionally used to study proteins, crowding inside cells can significantly alter the biophysical landscape of proteins, including their equilibrium thermodynamic stability (2)(3)(4)(5)(6). Experimental and computational efforts establish that crowding effects arise from a combination of short-range (steric) repulsions and longer-range (often called soft) interactions between macromolecules (7-13).…”

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confidence: 99%

Quinary structure modulates protein stability in cells

Monteith¹,

Cohen²,

Smith³

et al. 2015

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

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185

236

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Protein quinary interactions organize the cellular interior and its metabolism. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein-matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in the crowded cellular environment, but are traditionally studied in simple buffered solutions. We use NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli. We demonstrate that a surface mutation in this protein is 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions can be as large as the energies that stabilize specific protein complexes. These results will drive the critical task of implementing quinary structure into models for understanding the proteome.T he inside of cells is packed with macromolecules whose concentrations reach 300-400 g/L (1). Compared with the ideal (dilute) environments conventionally used to study proteins, crowding inside cells can significantly alter the biophysical landscape of proteins, including their equilibrium thermodynamic stability (2-6). Experimental and computational efforts establish that crowding effects arise from a combination of short-range (steric) repulsions and longer-range (often called soft) interactions between macromolecules (7-13). Despite a growing number of incell protein studies (2-6), there is no quantitative information about the energetics of quinary interactions.Amide proton exchange experiments have been used for more than 50 y to measure equilibrium protein stability, defined as the Gibbs free energy required to open the protein and expose individual backbone amide protons to solvent, ΔG°′ op (14). For the B1 domain of protein G (GB1), ΔG°′ op equals −RTln(k obs /k uns ), where R is the gas constant, T is the absolute temperature, k obs is the observed rate of exchange, and k uns is the rate in an unstructured peptide (6). We know that the cytoplasm does not affect k uns (15). Most importantly, we know that for exchange under these conditions ΔG°′ op approximates the free energy required to denature the protein, ΔG°′ den (6). Therefore, these experiments provide a thermodynamically rigorous measure of equilibrium global protein stability. Using this information, we quantified the stability of GB1 at the residue level in Escherichia coli (6) via NMRdetected backbone amide hydrogen/deuterium exchange (16).Thermodynamic cycles (17) can be used to quantify the energetics of interactions between proteins in specific protein complexes (17,18) and between side chains in globular proteins (19,20). Briefly, the individual effects of two single-site amino acid changes are compared with the combined effect of both mutations. If the sites interact, the sum of the contributions from the single-site changes will not equal the contributi...

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“…More recently, the same research group devised a way to obtain quantitative, residue-level information on the folding thermodynamics of intracellular GB1. The authors measured by NMR the amide hydrogen-deuterium exchange on cell lysates; after quenching by acidification, the hydrogen-deuterium exchange occurred in the cells at different times (42). With this approach, they showed that the cytoplasm stabilizes the folded state of GB1, whereas protein crowding agents in vitro have an opposite effect, thus highlighting the intrinsic complexity of the intracellular milieu.…”

Section: Effects Of Crowding On Folding and Weak Interactionsmentioning

confidence: 99%

A Unique Tool for Cellular Structural Biology: In-cell NMR

Luchinat

Banci

2016

Journal of Biological Chemistry

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Conventional structural and chemical biology approaches are applied to macromolecules extrapolated from their native context. When this is done, important structural and functional features of macromolecules, which depend on their native network of interactions within the cell, may be lost. In-cell nuclear magnetic resonance is a branch of biomolecular NMR spectroscopy that allows macromolecules to be analyzed in living cells, at the atomic level. In-cell NMR can be applied to several cellular systems to obtain biologically relevant structural and functional information. Here we summarize the existing approaches and focus on the applications to protein folding, interactions, and post-translational modifications.

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Residue level quantification of protein stability in living cells

Cited by 106 publications

References 72 publications

Equilibrium simulation of trp-cage in the presence of protein crowders

Equilibrium simulation of trp-cage in the presence of protein crowders

Quinary structure modulates protein stability in cells

A Unique Tool for Cellular Structural Biology: In-cell NMR

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