EGFP oligomers as natural fluorescence and hydrodynamic standards

György Vámosi,Katalin Tóth,Gabriele Müller,Jörg Langowski,Jan Wolfgang Krieger,Norbert Mücke,Ute Curth

doi:10.1038/srep33022

György Vámosi, Katalin Tóth + Show 5 more

Open Access

https://doi.org/10.1038/srep33022

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Abstract

EGFP oligomers are convenient standards for experiments on fluorescent protein-tagged biomolecules. In this study, we characterized their hydrodynamic and fluorescence properties. Diffusion coefficients D of EGFP1–4 were determined by analytical ultracentrifugation with fluorescence detection and by fluorescence correlation spectroscopy (FCS), yielding 83.4…48.2 μm2/s and 97.3…54.8 μm2/s from monomer to tetramer. A “barrels standing in a row” model agreed best with the sedimentation data. Oligomerization red-shifted EGFP emission spectra without any shift in absorption. Fluorescence anisotropy decreased, indicating homoFRET between the subunits. Fluorescence lifetime decreased only slightly (4%) indicating insignificant quenching by FRET to subunits in non-emitting states. FCS-measured D, particle number and molecular brightness depended on dark states and light-induced processes in distinct subunits, resulting in a dependence on illumination power different for monomers and oligomers. Since subunits may be in “on” (bright) or “off” (dark) states, FCS-determined apparent brightness is not proportional to that of the monomer. From its dependence on the number of subunits, the probability of the “on” state for a subunit was determined to be 96% at pH 8 and 77% at pH 6.38, i.e., protonation increases the dark state. These fluorescence properties of EGFP oligomeric standards can assist interpreting results from oligomerized EGFP fusion proteins of biological interest.

Highlights

Protein complexes in live cells are often investigated by observing fluorescent protein (FP) tags
For EGFP4 the Meq value was 10% smaller than Mcalc. This deviation and the systematic divergence of the fit residuals observed for EGFP4 (Supplementary Fig. S1) indicate an additional pool of smaller particles
Our group mapped the intracellular mobility of EGFP1–4 oligomers by single-point FCS5 and to study accessibility of nuclear regions

Summary

Introduction

Protein complexes in live cells are often investigated by observing fluorescent protein (FP) tags. Different photophysical processes of individual EGFP and other fluorescent proteins have already been described (photobleaching, triplet state formation, protonation- or light-induced “blinking”)[7,8,9,10,11,12,13]. There are still open questions how these processes contort the measured parameters (apparent brightness, apparent diffusion coefficient) of protein complexes containing more than one fluorescent protein. We test the effect of illumination power on the fluorescence parameters of the EGFP oligomers at two pH values and interpret how they relate to the behavior of the constituting monomeric subunits. We compare FCS-derived diffusion coefficients of EGFP oligomers with values yielded by analytical ultracentrifugation. We propose a simple procedure to quantify the probability of long-lived dark states of EGFP by analyzing the apparent brightness of the oligomers as a function of the number of subunits. DFCS0 was obtained from fits to FCS measurements, extrapolated to zero laser intensity

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