Ribosome Mediated Quinary Interactions Modulate In-Cell Protein Activities

DeMott, Christopher M.; Majumder, Subhabrata; Burz, David S.; Reverdatto, Sergey; Shekhtman, Alexander

doi:10.1021/acs.biochem.7b00613

Cited by 35 publications

(95 citation statements)

References 79 publications

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“…In vitro kinetic assays performed in the absence and presence of ribosomes and found that the ribosome acted as an uncompetitive inhibitor of ADK activity ( Figure 7A). Similar experiments performed using [U-2 D, 15 N] dihydrofolate reductase, DHFR, showed that DHFR engages in micromolar binding affinity quinary interactions with ribosomes, and that its coenzyme, NADPH, binds to ribosomes with a K d of ∼4.5 μM [10]. The results showed that the ribosome acts as a competitive inhibitor of DHFR activity ( Figure 7B).…”

Section: Functional Interactionssupporting

confidence: 65%

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Interaction proteomics by using in-cell NMR spectroscopy

Breindel

Burz

Shekhtman

2019

Journal of Proteomics

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The ultimate goal of structural and biochemical research is to understand how macromolecular interactions give rise to and regulate biological activity in living cells. The challenge is formidable due to the complexity that arises not only from the number of proteins (genes) expressed by the organism, but also from the combinatorial interactions between them. Despite ongoing efforts to decipher the complex nature of protein interactions, new methods for structurally characterizing protein complexes are needed to fully understand molecular networks. With the onset of in-cell NMR spectroscopy, molecular structures and interactions can be studied under physiological conditions shedding light on the structural underpinning of biological activity.

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Section: Functional Interactionssupporting

confidence: 65%

“…Excluded volume effects and molecular crowding increase the effective concentrations of cytosolic species, virtually assuring the likelihood of transient low-affinity interactions. In-cell NMR spectroscopy [7] has proven to be a reliable technique to identify protein interaction surfaces under these conditions [8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Interaction proteomics by using in-cell NMR spectroscopy

Breindel

Burz

Shekhtman

2019

Journal of Proteomics

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“…Recently, in‐cell NMR has emerged as a powerful technique for identifying sticking inside cells. In particular, these experiments have highlighted the importance of surface interactions in regulating non‐specific sticking of macromolecules, which can destabilize proteins, and suppress or activate enzymes . In vitro experiments in dilute cell lysates have had limited success replicating non‐steric interactions identified by in‐cell NMR …”

Section: Figurementioning

confidence: 99%

Non‐Steric Interactions Predict the Trend and Steric Interactions the Offset of Protein Stability in Cells

Davis

Gruebele

2018

ChemPhysChem

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Although biomolecules evolved to function in the cell, most biochemical assays are carried out in vitro. In-cell studies highlight how steric and non-steric interactions modulate protein folding and interactions. VlsE and PGK present two extremes of chemical behavior in the cell: the extracellular protein VlsE is destabilized in eukaryotic cells, whereas the cytoplasmic protein PGK is stabilized. VlsE and PGK are benchmarks in a systematic series of solvation environments to distinguish contributions from non-steric and steric interactions to protein stability, compactness, and folding rate by comparing cell lysate, a crowding agent, ionic buffer and lysate buffer with in-cell results. As anticipated, crowding stabilizes proteins, causes compaction, and can speed folding. Protein flexibility determines its sensitivity to steric interactions or crowding. Non-steric interactions alone predict in-cell stability trends, while crowding provides an offset towards greater stabilization. We suggest that a simple combination of lysis buffer and Ficoll is an effective new in vitro mimic of the intracellular environment on protein folding and stability.

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“…Non‐steric interactions refer to all interactions between the protein and its environment aside from volume‐excluding steric interactions, encompassing nonspecific sticking between macromolecules and weak quinary interactions evolved for function . The surface of proteins is particularly important in determining the strength of non‐steric interactions, which can stabilize or destabilize proteins and suppress or activate enzymes . To mimic non‐steric interactions in vitro, dilute cell lysate or lysis buffer can be added to the buffer .…”

Section: Introductionmentioning

confidence: 99%

An in vitro mimic of in‐cell solvation for protein folding studies

2020

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Ficoll, an inert macromolecule, is a common in vitro crowder, but by itself it does not reproduce in‐cell stability or kinetic trends for protein folding. Lysis buffer, which contains ions, glycerol as a simple kosmotrope, and mimics small crowders with hydrophilic/hydrophobic patches, can reproduce sticking trends observed in cells but not the crowding. We previously suggested that the proper combination of Ficoll and lysis buffer could reproduce the opposite in‐cell folding stability trend of two proteins: variable major protein‐like sequence expressed (VlsE) is destabilized in eukaryotic cells and phosphoglycerate kinase (PGK) is stabilized. Here, to discover a well‐characterized solvation environment that mimics in‐cell stabilities for these two very differently behaved proteins, we conduct a two‐dimensional scan of Ficoll (0–250 mg/ml) and lysis buffer (0–75%) mixtures. Contrary to our previous expectation, we show that mixtures of Ficoll and lysis buffer have a significant nonadditive effect on the folding stability. Lysis buffer enhances the stabilizing effect of Ficoll on PGK and inhibits the stabilizing effect of Ficoll on VlsE. We demonstrate that a combination of 150 mg/ml Ficoll and 60% lysis buffer can be used as an in vitro mimic to account for both crowding and non‐steric effects on PGK and VlsE stability and folding kinetics in the cell. Our results also suggest that this mixture is close to the point where phase separation will occur. The simple mixture proposed here, based on commercially available reagents, could be a useful tool to study a variety of cytoplasmic protein interactions, such as folding, binding and assembly, and enzymatic reactions. Significance Statement The complexity of the in‐cell environment is difficult to reproduce in the test tube. Here we validate a mimic of cellular crowding and sticking interactions in a test tube using two proteins that are differently impacted by the cell: one is stabilized and the other is destabilized. This mimic is a starting point to reproduce cellular effects on a variety of protein and biomolecular interactions, such as folding and binding.

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Ribosome Mediated Quinary Interactions Modulate In-Cell Protein Activities

Cited by 35 publications

References 79 publications

Interaction proteomics by using in-cell NMR spectroscopy

Interaction proteomics by using in-cell NMR spectroscopy

Non‐Steric Interactions Predict the Trend and Steric Interactions the Offset of Protein Stability in Cells

An in vitro mimic of in‐cell solvation for protein folding studies

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