Time-resolved two-photon fluorescence dynamics of a novel mEGFP-linker-mScarlet-I crowding biosensor.

Abstract

Laser-induced two-photon (2P) fluorescence microscopy is routinely used as a noninvasive, quantitative methodology for a wide range of cell and tissue studies due to its enhanced penetration depth, reduced laser scattering, and minimum extended photobleaching. Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Here, we carried out time-resolved 2P fluorescence measurements of a novel mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g/L) using Förster resonance energy transfer (FRET) analysis. For these studies, the excited-state 2P-fluorescence dynamics of the donor (mEGFP) were investigated in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet-I). In addition, time-resolved 2P-fluorescence measurements of intact GE2.3 were used to determine the corresponding equilibrium constant and the Gibbs free energy associated with the structural conformation equilibrium in response to environmental crowding. We further carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both the macromolecular crowding and the linker flexibility effects on GE2.3 rotational dynamics within the context of Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. Our results further the development of a rational engineering design of bioenvironmental sensors. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of crowded intracellular environment under different physiological conditions. Importantly, this GE2.3 sensor allows for minimal interference of the intrinsic cellular autofluorescence with the FRET analysis and crowding studies.