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 language||English (US)|
|Title of host publication||Ultrafast Nonlinear Imaging and Spectroscopy VII|
|Editors||Zhiwen Liu, Demetri Psaltis, Kebin Shi|
|State||Published - 2019|
|Event||Ultrafast Nonlinear Imaging and Spectroscopy VII 2019 - San Diego, United States|
Duration: Aug 11 2019 → Aug 12 2019
|Name||Proceedings of SPIE - The International Society for Optical Engineering|
|Conference||Ultrafast Nonlinear Imaging and Spectroscopy VII 2019|
|Period||8/11/19 → 8/12/19|
Bibliographical noteFunding 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.
© 2019 SPIE.
- Macromolecular crowding and ionic strength
- Time-resolved anisotropy
- Time-resolved fluorescence