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Tryptophan-based chromophore in fluorescent proteins can be anionic.

ABSTRACT: Cyan fluorescent proteins (CFP) with tryptophan66-based chromophore are widely used for live cell imaging. In contrast to green and red fluorescent proteins, no charged states of the CFP chromophore have been described. Here, we studied synthetic CFP chromophore and found that its indole group can be deprotonated rather easily (pKa 12.4).We then reproduced this effect in the CFP mCerulean by placing basic amino acids in the chromophore microenvironment. As a result, green-emitting variant with an anionic chromophore and key substitution Val61Lys was obtained. This is the first evidence strongly suggesting that tryptophan-based chromophores in fluorescent proteins can exist in an anionic charged state. Switching between protonated and deprotonated Trp66 in fluorescent proteins represents a new unexplored way to control their spectral properties.

SUBMITTER: Sarkisyan KS

PROVIDER: S-EPMC3429880 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Tryptophan-based chromophore in fluorescent proteins can be anionic.

Sarkisyan Karen S KS Yampolsky Ilia V IV Solntsev Kyril M KM Lukyanov Sergey A SA Lukyanov Konstantin A KA Mishin Alexander S AS

Scientific reports 20120829

Cyan fluorescent proteins (CFP) with tryptophan66-based chromophore are widely used for live cell imaging. In contrast to green and red fluorescent proteins, no charged states of the CFP chromophore have been described. Here, we studied synthetic CFP chromophore and found that its indole group can be deprotonated rather easily (pKa 12.4).We then reproduced this effect in the CFP mCerulean by placing basic amino acids in the chromophore microenvironment. As a result, green-emitting variant with a ...[more]

PMID: 22934131

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Project description:The chromophore of the green fluorescent protein (GFP) is critical for probing environmental influences on fluorescent protein behavior. Using the aqueous system as a bridge between the unconfined vacuum system and a constricting protein scaffold, we investigate the steric and electronic effects of the environment on the photodynamical behavior of the chromophore. Specifically, we apply ab initio multiple spawning to simulate five picoseconds of nonadiabatic dynamics after photoexcitation, resolving the excited-state pathways responsible for internal conversion in the aqueous chromophore. We identify an ultrafast pathway that proceeds through a short-lived (sub-picosecond) imidazolinone-twisted (I-twisted) species and a slower (several picoseconds) channel that proceeds through a long-lived phenolate-twisted (P-twisted) intermediate. The molecule navigates the non-equilibrium energy landscape via an aborted hula-twist-like motion toward the one-bond-flip dominated conical intersection seams, as opposed to following the pure one-bond-flip paths proposed by the excited-state equilibrium picture. We interpret our simulations in the context of time-resolved fluorescence experiments, which use short- and long-time components to describe the fluorescence decay of the aqueous GFP chromophore. Our results suggest that the longer time component is caused by an energetically uphill approach to the P-twisted intersection seam rather than an excited-state barrier to reach the twisted intramolecular charge-transfer species. Irrespective of the location of the nonadiabatic population events, the twisted intersection seams are inefficient at facilitating isomerization in aqueous solution. The disordered and homogeneous nature of the aqueous solvent environment facilitates non-selective stabilization with respect to I- and P-twisted species, offering an important foundation for understanding the consequences of selective stabilization in heterogeneous and rigid protein environments.

Dataset Information

Tryptophan-based chromophore in fluorescent proteins can be anionic.

Publications

Tryptophan-based chromophore in fluorescent proteins can be anionic.

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