Integrated fluorescence approach for FRET analysis of environmental sensors

Cody P. Aplin, Taryn M. Kay, Robert C. Miller, Arnold J. Boersma, Erin D. Sheets, Ahmed A. Heikal

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Förster resonance energy transfer (FRET) is considered as a molecular ruler to quantify protein-protein interactions and structural conformation in a wide range of biomolecules in both controlled environments and in living cells. Here, we have employed integrated fluorescence spectroscopy methods to characterize the energy transfer efficiency and donor-acceptor distance for novel genetically engineered mCerulean3-linker- mCitrine environmental sensors. Based on the amino acids sequences of the linker region, these sensors can be sensitive to either macromolecular crowding or the ionic strength of the surrounding environment. These hetero-FRET sensors also enable us to develop new spectroscopic approaches for quantifying the energy transfer efficiency and the donor-acceptor distance as a means of elucidating the underlying mechanisms for environmental sensing. Ensemble averaging approaches using time-resolved fluorescence and time-resolved fluorescence polarization anisotropy of G12 sensor are highlighted. Our findings in control environments so far are currently being used for complementary studies in living cells.

Original languageEnglish (US)
Title of host publicationUltrafast Nonlinear Imaging and Spectroscopy VII
EditorsZhiwen Liu, Demetri Psaltis, Kebin Shi
PublisherSPIE
ISBN (Electronic)9781510629370
DOIs
StatePublished - 2019
Externally publishedYes
EventUltrafast Nonlinear Imaging and Spectroscopy VII 2019 - San Diego, United States
Duration: Aug 11 2019Aug 12 2019

Publication series

NameProceedings of SPIE - The International Society for Optical Engineering
Volume11122
ISSN (Print)0277-786X
ISSN (Electronic)1996-756X

Conference

ConferenceUltrafast Nonlinear Imaging and Spectroscopy VII 2019
CountryUnited States
CitySan Diego
Period8/11/198/12/19

Bibliographical note

Funding Information:
We would like to thank Anh Cong, Elsie A. Johnson, and Christin Libal for their technical help and useful discussion during the course of this project. E.D.S. and A.A.H. acknowledge the financial support provided by the University of Minnesota Grant-in-Aid, a Chancellor’s Small Grant, the Department of Chemistry and Biochemistry, the Swenson College of Science and Engineering, University of Minnesota Duluth. A.J.B. acknowledges the financial support of the Netherlands Organization for Scientific Research Vidi grant. C.P.A. and R.C.M. were supported by teaching fellowships from the Department of Chemistry and Biochemistry, while T.M.K. was supported by a teaching fellowship from the Department of Physics and Astrophysics, University of Minnesota Duluth. We further acknowledge the support from the Minnesota Supercomputing Institute (MSI) at the University of Minnesota.

Publisher Copyright:
© 2019 SPIE.

Keywords

  • FRET
  • Macromolecular crowding and ionic strength
  • Mcerulean3
  • Mcitrine
  • Time-resolved anisotropy
  • Time-resolved fluorescence

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