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Live-cell and perfusion: weak protein-protein interactions

Weak protein-protein interaction in live-cells

Weak protein-protein interactions is a common phenomenon inside cells. Besides being well known, it has a crucial impact on cellular homeostasis and metabolism when involving key regulators. As examples, AMPK, PDK1, TOR or PGK are master regulators of cellular homeostasis as they all control key metabolic pathways. In this 2017 paper, Sukenic, Ren and Gruebele studied the impact of cell-volume on these protein aggregates looking at the specific case of GAPDH interaction with PGK. To achieve a fine control of the volume, the authors built a smart set-up to control the osmotic power of the media supply (see a comparison of perfusion methods here). Indeed, they realized a perfusion set-up allowing to switch between different media, all varing in osmotic pressure.
By further characterising the APDH-PGK complex association, Sukenic and co-authors suggested that cellular volume changes (as occuring when cells enter into mitosis), can be a regulatory mechanism controlling protein complex concentrations.

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Summary | Weakly bound protein complexes play a crucial role in metabolic, regulatory, and signaling pathways, due in part to the high tunability of their bound and unbound populations. This tunability makes weak binding (micromolar to millimolar dissociation constants) difficult to quantify under biologically relevant conditions. Here, we use rapid perturbation of cell volume to modulate the concentration of weakly bound protein complexes, allowing us to detect their dissociation constant and stoichiometry directly inside the cell. We control cell volume by modulating media osmotic pressure and observe the resulting complex association and dissociation by FRET microscopy. We quantitatively examine the interaction between GAPDH and PGK, two sequential enzymes in the glycolysis catalytic cycle. GAPDH and PGK have been shown to interact weakly, but the interaction has not been quantified in vivo. A quantitative model fits our experimental results with log Kd = −9.7 ± 0.3 and a 2:1 prevalent stoichiometry of the GAPDH:PGK complex. Cellular volume perturbation is a widely applicable tool to detect transient protein interactions and other biomolecular interactions in situ. Our results also suggest that cells could use volume change (e.g., as occurs upon entry to mitosis) to regulate function by altering biomolecular complex concentrations..

LEARN MORE | Shahar Sukenik et al. PNAS, 2017

Live-cell and perfusion : weak protein-protein interactions

Media osmotic pressures drives cells’ volume and consequent protein complexes bounding

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Long-term medium perfusion, change and drug injection

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