Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy
Author(s)Mofrad, Mohammad R.K.; Abdul Rahim, Nur Aida; Pelet, Serge; So, Peter T. C.; Kamm, Roger Dale
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Mechanical force modulates myriad cellular functions including migration, alignment, proliferation, and gene transcription. Mechanotransduction, the transmission of mechanical forces and its translation into biochemical signals, may be mediated by force induced protein conformation changes, subsequently modulating protein signaling. For the paxillin and focal adhesion kinase interaction, we demonstrate that force-induced changes in protein complex conformation, dissociation constant, and binding Gibbs free energy can be quantified by lifetime-resolved fluorescence energy transfer microscopy combined with intensity imaging calibrated by fluorescence correlation spectroscopy. Comparison with in vitro data shows that this interaction is allosteric in vivo. Further, spatially resolved imaging and inhibitor assays show that this protein interaction and its mechano-sensitivity are equal in the cytosol and in the focal adhesions complexes indicating that the mechano-sensitivity of this interaction must be mediated by soluble factors but not based on protein tyrosine phosphorylation.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Mechanical Engineering; Massachusetts Institute of Technology. Laser Biomedical Research Center
Abdul Rahim, Nur Aida; Pelet, Serge; Mofrad, Mohammad R.K.; So, Peter T.C. and Kamm, Roger D. “Quantifying Intracellular Protein Binding Thermodynamics During Mechanotransduction Based on FRET Spectroscopy.” Methods 66, no. 2 (March 2014): 208–221 © 2013 Published by Elsevier Inc
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