Rational design of a pH‐insensitive cyan fluorescent protein CyPet2 based on the CyPet crystal structure

Abstract

The emission spectrum of widely used CyPet is pH-sensitive. In order to synthesize a pH-insensitive cyan fluorescent protein by rational design, we solved the crystal structures of CyPet under different pH conditions. The indole group of the CyPet chromophore adopts a cis-coplanar conformation in acidic and neutral conditions, while it converts to trans-coplanar under basic conditions. His148 and Glu222 play a vital role in this isomerization. The pH-sensitive chromophore isomerization and change in the emission spectrum can be explained by the coexistence of several different fluorescent states. We trap the chromophore in the trans conformation by A167I mutation (CyPet2), which also prevents the multiconformation of the seventh β-strand. CyPet2 exhibits an unchanged emission spectral shape as a function of pH.

Cyan fluorescent proteins derived from mNeonGreen

“…The Z , E configuration has consistently been associated with a blue-shifted spectral profile for the neutral chromophore ( Gotthard et al. 2017 ; Liu et al. 2017 ; Malo et al.…”

“…2007 ), SCFP3A at pH 4.5 ( Gotthard et al. 2017 ), CyPet at pH < 7.0 ( Liu et al. 2017 ), and NowGFP at pH 4.8 (20% Z , E and 80% Z,Z ) ( Pletnev et al.…”

Chromophore Isomer Stabilization Is Critical to the Efficient Fluorescence of Cyan Fluorescent Proteins

“…This can be explained by the fact that residue 148 can be a H-bond acceptor in Cerulean and SCFP3A because of the six lone pairs of a deprotonated aspartate (p K a = 3.9), but not in ECFP because a doubly protonated histidine (p K a = 6.1) does not have any lone pair. CyPet also has a histidine at position 148, and the Z , E isomer seen in its structure obtained at pH 7.0 can be rationalized by the fact that His148 is still singly protonated at this pH and as such can provide a lone pair to stabilize the indole ring of the chromophore.…”

“…The structure shows a chromophore in the Z , E configuration with interactions with neighboring residues similar to those in Cerulean, except that there is only one H bond between Asp148 and the chromophore and Ser205 is fully rotated away from Glu222 (Figure B, left). The last of the second-generation CFPs, CyPet, has recently been shown to undergo the same pH-induced isomerization, yet with a much higher p K a (>7.0) …”

“…The last of the second-generation CFPs, CyPet, has recently been shown to undergo the same pH-induced isomerization, yet with a much higher pK a (>7.0). 20 Visualizing the pH-induced isomerization in all secondgeneration CFPs raised the question of whether this phenomenon also occurs in their precursor ECFP. pH titration of absorption properties of Cerulean and SCFP3A from pH 8.0 to 3.0 reveals a blue shift before acidic denaturation (Figure 1C) characteristic of the chromophore isomerization.…”