Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer

Abstract

Fö rster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (hE exp FRET i ¼ 0.25 5 0.05) and those calculated from the molecular dynamics simulations (hE MD FRET i ¼ 0.18 5 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors hk 2 i ¼ 0.17 5 0.16 and hjk j i ¼ 0.35 5 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time-and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET.

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

“…We note that the observed reduction in mGFP lifetime was approximately 10-fold lower than that observed in the case of the crGE2.3 FRET. This reduction is likely due to a change in refractive index when glycerol is introduced, which has been shown to decrease fluorescence lifetimes of not only GFP [63][64][65][66][67] but other fluorophores and fluorescent proteins such as fluorescein, CFP and YFP [68,69]. Previous studies also show that Ficoll 70 and Ficoll 400 increase the refractive index of solution [51] suggesting that the reduction in lifetime seen for all molecular crowders used here could be due to refractive index changes.…”

Extending fluorescence anisotropy to large complexes using reversibly switchable proteins

“…Because the kinetics of ON-switching are much more rapid than OFF-switching in rsEGFP2 15 , 16 and Dronpa 14 , this method is the most sensitive to fast rotational diffusion. We observed rotational diffusivity of molecules as small as rsEGFP2 (∼16.5 ns of EGFP) 17 .…”

Influence of graphene oxide on the bile salts–ligand interaction: a spectroscopy study

“…The rotational correlational time is defined as the time required by the fluorophore to rotate 1 radian. 63 Hence, time-resolved fluorescence anisotropy is a powerful tool to investigate the microenvironment surrounding the fluorophore molecule. In essence, it can monitor the restriction of the rotational mobility of the NB molecules in different systems, since one of the major inferences drawn from anisotropy data is the degree of restriction that the fluorophore under investigation experiences in different environments.…”