Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools

Yi Cui; Samrat Roy Choudhury; Joseph Irudayaraj

doi:10.1016/j.ab.2014.04.023

Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools

Yi Cui, Samrat Roy Choudhury, Joseph Irudayaraj

Research output: Contribution to journal › Article › peer-review

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 300 s detection window.

Original language	English (US)
Pages (from-to)	58-60
Number of pages	3
Journal	Analytical Biochemistry
Volume	458
DOIs	https://doi.org/10.1016/j.ab.2014.04.023
State	Published - Aug 1 2014
Externally published	Yes

Keywords

CIB1
CIBN
CRY2
FCS
FRET
Optogenetic protein

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

Online availability

10.1016/j.ab.2014.04.023

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title = "Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools",

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 F{\"o}rster 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 300 s detection window.",

keywords = "CIB1, CIBN, CRY2, FCS, FRET, Optogenetic protein",

author = "Yi Cui and Choudhury, {Samrat Roy} and Joseph Irudayaraj",

note = "Funding Information: We thank Dr. Xiaolei Wang for the help in optical instrumentation. Support from the W.M. Keck Foundation (United States) grant and the Purdue Center for Cancer Research through the innovative grant are appreciated. ",

year = "2014",

month = aug,

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doi = "10.1016/j.ab.2014.04.023",

language = "English (US)",

volume = "458",

pages = "58--60",

journal = "Analytical Biochemistry",

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T1 - Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools

AU - Cui, Yi

AU - Choudhury, Samrat Roy

AU - Irudayaraj, Joseph

N1 - Funding Information: We thank Dr. Xiaolei Wang for the help in optical instrumentation. Support from the W.M. Keck Foundation (United States) grant and the Purdue Center for Cancer Research through the innovative grant are appreciated.

PY - 2014/8/1

Y1 - 2014/8/1

N2 - 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 300 s detection window.

AB - 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 300 s detection window.

KW - CIB1

KW - CIBN

KW - CRY2

KW - FCS

KW - FRET

KW - Optogenetic protein

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SN - 0003-2697

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JO - Analytical Biochemistry

JF - Analytical Biochemistry

ER -

Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools

Abstract

Keywords

ASJC Scopus subject areas

Online availability

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