Paper

Macromolecular crowding effects on energy transfer efficiency and donor-acceptor distance of hetero-FRET sensors using time-resolved fluorescence

, , , , , , and

Published 19 February 2019 © 2019 IOP Publishing Ltd
, , Citation Jacob Schwarz et al 2019 Methods Appl. Fluoresc. 7 025002 DOI 10.1088/2050-6120/ab0242

2050-6120/7/2/025002

Abstract

Living cells are crowded with macromolecules and organelles, which affect a myriad of biochemical processes. As a result, there is a need for sensitive molecular sensors for quantitative, site-specific assessment of macromolecular crowding. Here, we investigated the excited-state dynamics of recently developed hetero-FRET sensors (mCerulean3–linker–mCitrine) in homogeneous and heterogeneous environments using time-resolved fluorescence measurements, which are compatible with fluorescence lifetime imaging microscopy (FLIM). The linker in these FRET constructs, which tether the mCerulean3 (the donor) and mCitrine (the acceptor), vary in both length and flexibility. Glycerol and Ficoll-70 solutions were used for homogeneous and heterogeneous environments, respectively, at variable concentrations. The wavelength-dependent studies suggest that the 425-nm excitation and the 475-nm emission of the donor are best suited for quantitative assessment of the energy transfer efficiency and the donor-acceptor distance of these FRET probes. Under the same experimental conditions, the enzymatically cleaved counterpart of these probes was used as a control as well as a means to account for the changes in the environmental refractive indices. Our results indicate that the energy transfer efficiency of these FRET probes increases as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor a compact structure with enhanced energy transfer efficiency and a shorter donor-acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the stretched conformation of these FRET probes is more favorable in the viscous, homogeneous environment with a reduced energy transfer efficiency and relatively larger donor-acceptor distance as compared with those in pure buffer, which was attributed to a reduced structural fluctuation of the mCerulean3–mCitrine FRET pair in the viscous, more restrictive glycerol-enriched buffer. Our findings will help to advance the potential of these hetero-FRET probes using FLIM for spatio-temporal assessment of the compartmentalized crowding in living cells.

Export citation and abstract BibTeX RIS

Access this article

The computer you are using is not registered by an institution with a subscription to this article. Please choose one of the options below.

Login

IOPscience login

Find out more about journal subscriptions at your site.

Purchase from

Article Galaxy
CCC RightFind

Purchase this article from our trusted document delivery partners.

Make a recommendation

To gain access to this content, please complete the Recommendation Form and we will follow up with your librarian or Institution on your behalf.

For corporate researchers we can also follow up directly with your R&D manager, or the information management contact at your company. Institutional subscribers have access to the current volume, plus a 10-year back file (where available).

Please wait… references are loading.
10.1088/2050-6120/ab0242