Examining the Donor Effect on the Environmental Sensitivity of Macromolecular Crowding Sensors (2022-03-18)

Abstract

Macromolecular crowding influences many biological processes in living cells such as protein folding, protein-protein interactions, translational diffusion, and biochemical reaction kinetics. The challenge, however, is that environmental parameters are heterogeneous, dynamic, and sensitive to the biology of the cell. Past studies have relied on invasive techniques that destroy the cell microenvironment and are currently incompatible with leading-edge biochemical research. Here, we investigate a family of environmental biosensors made of donor–

linker–acceptor constructs using quantitative, noninvasive laser-induced, time- resolved two-photon fluorescence. In these constructs, the donor-acceptor pairs are

engineered using cyan and yellow fluorescent proteins with a neutral double helix linker. These intrinsically fluorescent proteins were selected based on their ability to undergo Förster resonance energy transfer (FRET) as an indicator for conformational changes in response to environmental factors. We investigated the effects of donor identity (mCerulean3, mTurqoise2.1, and mTurqoise2.0) on the environmental sensitivity of these biosensors. Our hypothesis is that the sensors with turquoise variants as FRET donors will exhibit a higher energy transfer efficiency (i.e., higher environmental sensitivity) as compared with the mCerulean3 construct due to their enhanced spectral overlap, excitation cross-section, and fluorescence quantum yield. We also carried out comprehensive control experiments on enzymatically cleaved sensors (i.e., donor alone) under identical experimental conditions. An additional control was carried out in glycerol-enriched buffer as a means to differentiate between crowding and viscosity effects on the observed FRET. These studies in controlled environments are essential for (i) developing rational design strategies of new and optimized environmental biosensors, and (ii) exploiting the advantages of nonlinear laser spectroscopy in future biological cells expressing these biosensors for diagnostic purposes.