Chromophore Environment Provides Clue to “Kindling Fluorescent Protein” Riddle

Dmitriy M Chudakov,Alexei V Feofanov,Nikolay N Mudrik,Sergey Lukyanov,Konstantin A Lukyanov

doi:10.1074/jbc.m211988200

Abstract

asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

Highlights

Escherichia coli and purified using the Talon metal affinity resin (Clontech)
Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr66 in the chromophore are crucial for kindling
Concluding Comments—The kindling effect was relatively easy to transfer to nonkindling, nonfluorescent, GFP-like Anthozoa chromoproteins

Summary

Introduction

Escherichia coli and purified using the Talon metal affinity resin (Clontech). Site-directed mutagenesis was performed by overlap extension PCR with primers containing the appropriate target substitutions [2]. AsCP becomes fluorescent (kindles) in response to intense green light irradiation, with an excitation maximum at 575 nm and an emission maximum at 595 nm. These data can be used as three-dimensional models and are suitable for the analysis of asCP and other GFP-like fluorescent proteins (FPs) and nonfluorescent chromoproteins (CPs).

Results

Conclusion