Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy
Name
Kamm_Quantifying intracellular.pdf
Size
905.72 KB
Format
Adobe PDF
Checksum (MD5)
ca367a473638e5106fcfe83500d55fd3
Author(s)
Mofrad, Mohammad R.K.
Abdul Rahim, Nur Aida
Pelet, Serge
So, Peter T. C.
Kamm, Roger Dale
Date Issued
October 2013
Journal
Methods
Publisher
Elsevier
Citation
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
Version
Author's final manuscript
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Mechanical Engineering
Massachusetts Institute of Technology. Laser Biomedical Research Center
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1016/j.ymeth.2013.10.007
