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Photophysical Behavior of mNeonGreen, an Evolutionarily Distant Green Fluorescent Protein

MPS-Authors
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Steiert,  Frederik
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Petrov,  Eugene P.
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Schultz,  Peter
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Schwille,  Petra
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Weidemann,  Thomas
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Steiert, F., Petrov, E. P., Schultz, P., Schwille, P., & Weidemann, T. (2018). Photophysical Behavior of mNeonGreen, an Evolutionarily Distant Green Fluorescent Protein. Biophysical Journal, 114(10), 2419-2431. doi:10.1016/j.bpj.2018.04.013.


Cite as: http://hdl.handle.net/21.11116/0000-0002-C912-7
Abstract
Fluorescent proteins (FPs) feature complex photophysical behavior that must be considered when studying the dynamics of fusion proteins in model systems and live cells. In this work, we characterize mNeonGreen (mNG), a recently introduced FP from the bilaterian Branchiostoma lanceolatum, in comparison to the well-known hydrozoan variants enhanced green fluorescent protein (EGFP) and Aequorea coerulescens GFP by steady-state spectroscopy and fluorescence correlation spectroscopy in solutions of different pH. Blind spectral unmixing of sets of absorption spectra reveals three interconverting electronic states of mNG: a nonfluorescent protonated state, a bright state showing bell-shaped pH dependence, and a similarly bright state dominating at high pH. The gradual population of the acidic form by external protonation is reflected by increased flickering at low pH in fluorescence correlation spectroscopy measurements, albeit with much slower flicker rates and lower amplitudes as compared to Aequorea GFPs. In addition, increased flickering of mNG indicates a second deprotonation step above pH 10 leading to a slight decrease in fluorescence. Thus, mNG is distinguished from Aequorea GFPs by a two-step protonation response with opposite effects that reflects a chemically distinct chromophore environment. Despite the more complex pH dependence, mNG represents a superior FP under a broad range of conditions.