Time-Resolved Anisotropy and Conformational Analysis of Hetero-FRET Environmental Biosensors Undergoing Two-Photon Excitation (2022-04-01)

Abstract

Compartmentalized, dynamic ionic strength within living cells influences numerous biochemical mechanisms such as catalytic function, protein folding, osmotic pressure, and energy production. The recent development of genetically encoded biosensors that undergo Förster resonance energy transfer (FRET) offers a promising methodology towards noninvasive, site-specific, quantitative, and sensitive mapping of in vivo ionic strength. Our sensors consist of a flexible linker made of one (K6 sensor) or two (RD sensor) oppositely charged alpha helices, which separates a cyan fluorescent protein (mCerulean3 or mTurqoise2.1) donor and a yellow fluorescent protein (mCitrine) acceptor. In this project, we investigate the effects of the amino acid sequence in the linker region on the sensitivity to environmental ionic strength. Towards that goal, we developed a new time-resolved two-photon (2P) fluorescence depolarization approach for FRET analysis, where the donor is excited by 850–nm polarized laser pulses, and the polarized parallel and perpendicular emission from the acceptor is simultaneously detected. As controls, donor–linker–acceptor constructs with electrostatically neutral alpha helices (E6G2 and E6) and enzymatically cleaved sensors (i.e., the donor alone) are measured under the same experimental conditions. Our results show that as ionic strength increased, the donor-acceptor distance increased due to electrostatic screening of the helix charges, resulting in reduced FRET efficiency. Additionally, our experimental data was modeled to quantify the conformational changes in the population fractions of collapsed (FRETing) and stretched (non–FRETing) constructs to calculate the equilibrium constant and corresponding Gibbs free energy. These results show an increase in the fraction of stretched constructs and a decrease of Gibbs free energy as a function of ionic strength. This study has taken an important step toward future in vivo studies by further developing the rational design of the biosensors as well as optimizing techniques of experimentation and methodology of FRET analysis.