Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools.
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.
Department of Agricultural and Biological Engineering, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, United States.
Yi
Y
Cui
Department of Agricultural and Biological Engineering, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, United States.
Samrat Roy
SR
Choudhury
Department of Agricultural and Biological Engineering, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, United States. Electronic address: josephi@purdue.edu.
Joseph
J
Irudayaraj
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D056931:Cryptochromes / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D056931:Cryptochromes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C533832:CIB1 protein, Arabidopsis; C103618:CRY2 protein, Arabidopsis
CIB1; CIBN; CRY2; FCS; FRET; Optogenetic protein