Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB193

Poster Communications

Detection of intracellular cysteine modification with green fluorescent protein variants

J. M. Langner1, I. Coburger1, N. Kuldyushev1, K. Yang1, R. Schönherr1, S. H. Heinemann1

1. Department of Biophysics, Friedrich-Schiller-University Jena, Jena, Germany.

Post-translational protein modifications at cysteine residues play key roles in regulatory processes, and excess cysteine oxidation may induce protein damage and contributes to degenerative phenomena. Commonly applied methods for the detection of thiol modification in intracellular proteins involve their biochemical workup. Genetically encoded redox-sensitive fluorescence indicators hold promise as real-time sensors for live-cell imaging with minimal impact on the natural cellular state (1). The redox-sensitive green fluorescent protein (GFP) variant roGFP2 is a powerful tool for studying formation and breakage of a disulphide bridge in living cells (2). In roGFP2 one endogenous cysteine is eliminated (C48S), and two are introduced in the neighbourhood of the chromophore (S147C, Q204C), such that they are accessible and close enough to form a disulphide bridge upon oxidation. Subsequent structural rearrangement leads to an increase of fluorescence intensity at 400 nm and a decrease in intensity at 470 nm, thus providing a ratiometric signal related to the redox state (F400/F470 = r). A roGFP2 signal, however, does not provide information on the type of modification that occurred at either one of the two cysteine residues. We therefore examined the properties of the individual cysteine mutants (S147C, Q204C) by performing real-time fluorescence imaging 24 h after transfection in HEK293T cells (21-24 °C). In resting cells, the following F400/F470 were determined (mean ± sem, n = number of cells): roGFP2, 0.024 ± 0.0005 (n = 208); S147C, 0.027 ± 0.0004 (n = 175); Q204C, 0.036 ± 0.0003 (n = 140). The time course of r was recorded in response to extracellularly applied cysteine modifying agents. 2,2'-Dithio-bis(5-Nitropyridine) (DTNP, 20 µM), a specific Cys modifier, elicited an increase in F400/F470 by 0.09 ± 0.0005 with an exponential time constant of 46.7 ± 1.12 s (n = 24). The ratio for mutant S147C increased by 0.02 ± 0.004 (tau = 995 ± 227 s, n = 28) whereas mutant Q204C responded with a decrease in r: Δr = -0.01 ± 0.0002 (tau = 119 ± 4.4 s, n = 20). Exposure of the cells to diamide (100 µM), which is expected to mediate glutathionylation of cysteines (3), resulted in the following changes: roGFP2, Δr = 0.10 ± 0.002, tau = 31.2 ± 1.12 s, (n = 24); S147C, Δr = 0.01 ± 0.0007, tau = 319 ± 47 s, (n = 24); Q204, Δr = -0.01 ± 0.00009, tau = 252 ± 6.3 s, (n = 18). Application of Na2S4 (10 µM), a polysulfide donor that is expected to induce cysteine sulfhydration, also affected r for roGFP2: Δr = 0.06 ± 0.001, tau = 148 ± 7.4 s (n = 23), while mutant S147C (n = 21) only showed a slight increase in r, and no response of Q204C (n = 16) was detected. Cysteines at positions 147 and 204 in GFP have the potential of reporting on intracellular thiol modification by providing ratiometric fluorescence signals.

Where applicable, experiments conform with Society ethical requirements