Wavelength-dependent fluorescence fluctuation and molecular brightness analysis of a novel mEGFP-linker-mScarlet-I crowding biosensor at the single-molecule level.

Abstract

Living cells are dynamic and heterogeneous microenvironments, in which macromolecular crowding impacts transport, intermolecular interactions, and kinetics. Here, we characterize a protein biosensor that has been designed to detect changes in macromolecular crowding using Förster energy transfer (FRET). We investigate the fluorescence fluctuations, molecular brightness, and translational diffusion of the mEGFP-linker-mScarlet-I construct (GE2.3) using fluorescence correlation spectroscopy (FCS) of the donor (mEGFP) in the presence and absence of the acceptor (mScarlet-I) in response to environmental crowding. Our FCS setup was calibrated using rhodamine-110, and control experiments were also carried out on the enzymatically cleaved GE2.3 to elucidate the effects of FRET on the molecular brightness at the single molecule level. Our results indicate that the molecular brightness of intact GE2.3 (PBS buffer, pH 7.4) is smaller than that of the cleaved counterpart under 488-nm excitation of the donor, due to FRET. In contrast, the molecular brightness of both cleaved and intact GE2.3 seems to be the same under the 561-nm excitation of the acceptor due to the absence of FRET. The fluctuation analyses of intact and cleaved GE2.3 sensors were also used to investigate the crowding effects on translational diffusion within the context of Stokes-Einstein model. Our single-molecule findings for FRET analysis of GE2.3 complements our ensemble studies using time-resolved fluorescence and anisotropy (rotational depolarization dynamics) because it allows us to discern the temporal-scale dependence (rotational versus translational diffusion) of potential segmental mobility of the construct as a function of crowding. Further, these studies are helping to develop a rational design strategy for environmental sensors by examining different donor-acceptor FRET pair, while refining the use of donor molecular brightness to measure FRET at the single-molecule level using a traditional FCS setup.