Abstract
Biochemistry in living cells is an emerging field of science. Current quantitative bioassays are performed ex vivo, thus equilibrium constants and reaction rates of reactions occurring in human cells are still unknown. To address this issue, we present a non-invasive method to quantitatively characterize interactions (equilibrium constants, KD) directly within the cytosol of living cells. We reveal that cytosolic hydrodynamic drag depends exponentially on a probe’s size, and provide a model for its determination for different protein sizes (1–70 nm). We analysed oligomerization of dynamin-related protein 1 (Drp1, wild type and mutants: K668E, G363D, C505A) in HeLa cells. We detected the coexistence of wt-Drp1 dimers and tetramers in cytosol, and determined that KD for tetramers was 0.7 ± 0.5 μM. Drp1 kinetics was modelled by independent simulations, giving computational results which matched experimental data. This robust method can be applied to in vivo determination of KD for other protein-protein complexes, or drug-target interactions.
Highlights
Biomolecular interactions are the basic components of life processes[1]
The hydrodynamic drag of cytoplasm in HeLa cells depends on length scale
We discussed quantitative interpretation of diffusion coefficients recorded by means of FCS in cytosol of living cells
Summary
Biomolecular interactions are the basic components of life processes[1]. Cellular metabolism, function, division, and fate rely on a network of interconnected biochemical reactions, and any disruption of this fragile balance can lead to pathological changes in the cell or a whole organism. Many advanced methods have been developed to identify and quantify biomolecular interactions in living cells[3,4], but most approaches (e.g. mass spectrometry, yeast two-hybrid system, confocal imaging) concerning in situ interactions are qualitative or semi-quantitative[5] Quantitative assays such as surface plasmon resonance and biochemical tests require purified and processed samples (by fixation or extraction of the material)[4], which prohibits their application to living cells. The FRET signal’s presence and change over time can be interpreted in terms of reaction kinetics[7,9] This technique has so far been applied to the binding of bacterial proteins[7], conformational changes of a human protein[8] in HeLa cells, and the interaction between an enzyme and a product in HEK293T cells[9]. All parameters of Eq 1 depend on cell type and cell culture conditions[21]; every experiment, in which cytoplasmic viscosity is a parameter, should be preceded with careful determination of ηeff at the length scale of interest
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