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Article Dans Une Revue Journal of the American Chemical Society Année : 2021

Disentangling Chromophore States in a Reversibly Switchable Green Fluorescent Protein: Mechanistic Insights from NMR Spectroscopy

Résumé

The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its p-HBI chromophore, corresponding to the cis and trans isomers, with each one either protonated (neutral) or deprotonated (anionic) at the benzylidene ring. The relative populations and interconversion kinetics of these chromophore species depend on sample pH and buffer composition that alter in a complex way the strength of H-bonds that contribute in stabilizing the chromophore within the protein scaffold. We show in particular the important role of histidine-149 in stabilizing the neutral trans chromophore at intermediate pH values, leading to ground-state cis-trans isomerization with a peculiar pH dependence. We discuss the potential implications of our findings on the pH dependence of the photoswitching contrast, a critical parameter in nanoscopy applications.
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hal-03328569 , version 1 (30-08-2021)

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Nina Eleni Christou, Karine Giandoreggio-Barranco, Isabel Ayala, Oleksandr Glushonkov, Virgile Adam, et al.. Disentangling Chromophore States in a Reversibly Switchable Green Fluorescent Protein: Mechanistic Insights from NMR Spectroscopy. Journal of the American Chemical Society, 2021, 143 (19), pp.7521-7530. ⟨10.1021/jacs.1c02442⟩. ⟨hal-03328569⟩
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