Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools
Yi Cui1, Samrat Roy Choudhury1, Joseph Irudayaraj1
1Department of Agricultural and Biological Engineering, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, United States.
Abstract:
In this work we evaluate the interaction of two optogenetic protein variants (CIB1, CIBN) with their complementary protein CRY2 by single-molecule tools in cell-free extracts. After validating the blue light induced co-localization of CRY2 and CIB1/N by Förster resonance energy transfer (FRET) in live cells, a fluorescence correlation spectroscopy (FCS) based method was developed to quantitatively determine the in vitro association of the extracted proteins. Our experiments suggest that CIB1, in comparison with CIBN, possesses a better coupling efficiency with CRY2 due to its intact protein structure and lower diffusion rate within 300s detection window.


