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

Abstract

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 4 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.

Protonation States of Molecular Groups in the Chromophore-Binding Site Modulate Properties of the Reversibly Switchable Fluorescent Protein rsEGFP2

“…When studying a closely related protein rsFolder, Cristou et al 12 concluded that solvent protons can easily migrate to the chromophore-containing pocket and protonate the hydroxybenzylidene ring of the chromophore "on a submilliseconds time scale". In line with this finding, which presumably holds for rsEGFP2 as well, the following equilibrium is addressed by our models of the ON-state (A-cis)  + H +  (N-cis)  (Eq.…”

“…5 The Chro protonation state alteration as well as of the surrounding Chro molecular groups was also put forward by studies of the OFF → ON transformations of rsEGFP2. 7,8 When discussing protonation in rsEGFP2, we note the very recent paper 12 that reports results of the NMR studies for a related GFP-like reversibly switchable protein rsFolder with the same composition of the chromophore and only few variations in amino acid residues neighboring to the chromophore. 6 The obtained data show that the relative population of the chromophore ON-and OFFstates vary in a complex pH-dependent manner.…”

“…The protonated and deprotonated forms of Chro and nearby residues play an important role in hydrogen-bond dynamics, altering the relative stability of the cis and trans configurations of Chro. Protonation-dependent dynamics determines the rsFolder contrast, 12 an important parameter in applications of the reversibly switchable fluorescent probe. 5 Therefore, the distinct properties of the cis and trans forms of Chro revealed in recent experimental studies of rsEGFP2 and rsFolder [5][6][7][8]12 are linked to a specific location of protons at various molecular groups in the chromophore-binding sites.…”

“…Protonation-dependent dynamics determines the rsFolder contrast, 12 an important parameter in applications of the reversibly switchable fluorescent probe. 5 Therefore, the distinct properties of the cis and trans forms of Chro revealed in recent experimental studies of rsEGFP2 and rsFolder [5][6][7][8]12 are linked to a specific location of protons at various molecular groups in the chromophore-binding sites. This knowledge is indispensable to understand mechanistic details of photoswitching.…”

Protonation States of Molecular Groups in the Chromophore-Binding Site Modulate Properties of the Reversibly Switchable Fluorescent Protein rsEGFP2

“…5 The Chro protonation state alteration as well as of the surrounding Chro molecular groups was also put forward by studies of the OFF → ON transformations of rsEGFP2. 7,8 When discussing protonation in rsEGFP2, we note the very recent paper 12 that reports results of the NMR studies for a related GFP-like reversibly switchable protein rsFolder with the same composition of the chromophore and only few variations in amino acid residues neighboring to the chromophore. 6 The obtained data show that the relative population of the chromophore ON-and OFFstates vary in a complex pH-dependent manner.…”