TY - JOUR
T1 - Weak protein-protein interactions in live cells are quantified by cell-volume modulation
AU - Sukenik, Shahar
AU - Ren, Pin
AU - Gruebele, Martin
N1 - Funding Information:
We thank A. L. DeVries for use of his osmometer and E. L. Snapp for helpful discussions and the kind donation of the mEGFP plasmid. We acknowledge funding from National Science Foundation Grant MCB14-13256 for development of the volume-jump microscopy and National Institutes of Health Grant GM093318-01-08 for in vitro association measurements.
PY - 2017/6/27
Y1 - 2017/6/27
N2 - 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.
AB - 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.
KW - Cell volume
KW - Fret
KW - Live-cell microscopy
KW - Protein-protein interactions
KW - Quinary interactions
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U2 - 10.1073/pnas.1700818114
DO - 10.1073/pnas.1700818114
M3 - Article
C2 - 28607089
AN - SCOPUS:85021415002
SN - 0027-8424
VL - 114
SP - 6776
EP - 6781
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 26
ER -