rXXXX American Chemical Society A dx.doi.org/10.1021/jp111593x | J. Phys. Chem. B XXXX, XXX, 000–000 ARTICLE pubs.acs.org/JPCB Chemically Modulating the Photophysics of the GFP Chromophore Jamie Conyard, † Minako Kondo, †,§ Ismael A. Heisler, † Garth Jones, † Anthony Baldridge, ‡ Laren M. Tolbert, ‡ Kyril M. Solntsev,* ,‡ and Stephen R. Meech* ,† † School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom ‡ School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Dr., 30332-0400, United States b S Supporting Information ABSTRACT: There is growing interest in engineering the properties of fluorescent proteins through modifications to the chromophore structure utilizing mutagenesis with either natural or unnatural amino acids. This entails an under- standing of the photophysical and photochemical properties of the modified chromophore. In this work, a range of GFP chromophores with different alkyl substituents are synthe- sized and their electronic spectra, pH dependence, and ultrafast fluorescence decay kinetics are investigated. The weakly electron donating character of the alkyl substituents leads to dramatic red shifts in the electronic spectra of the anions, which are accompanied by increased fluorescence decay times. This high sensitivity of electronic structure to substitution is also characteristic of some fluorescent proteins. The solvent viscosity dependence of the decay kinetics are investigated, and found to be consistent with a bimodal radiationless relaxation coordinate. Some substituents are shown to distort the planar structure of the chromophore, which results in a blue shift in the electronic spectra and a strong enhancement of the radiationless decay. The significance of these data for the rational design of novel fluorescent proteins is discussed. ’ INTRODUCTION The green fluorescent protein is established as a key tool in life sciences. 1 The relative ease with which GFP can be cloned and expressed, combined with the intense green fluorescence from its uniquely covalently bound fluorophore, permits 3D spatial resolution of specifically labeled proteins in living organisms. 2,3 From this original protein, a family of chromo- proteins has been developed either through discovery or mutag- enesis or, more recently, exploitation of non-natural amino acids. 4-10 These proteins share the same basic β-barrel struc- ture as GFP with only minor modifications in the chromophore and its environment, but nevertheless exhibit an enormous variety of photophysical properties which are revolutionizing bioimaging. Niwa and co-workers first identified the GFP chromophore and synthesized its close analogue p-hydroxybenzilideneimida- zolinone (HBDI, Figure 1). 11,12 Because of the wide importance of GFP fluorescence, the spectroscopy and photophysics of HBDI have attracted considerable attention. 13,14 Niwa et al. 12 correlated the electronic spectra of the chromophore with that of the protein, while others 15-17 used Raman spectroscopy to confirm the charge state of the chromophore in the protein. One unexpected feature of HBDI photophysics is that its fluorescence in fluid solvents is extremely weak—a quantum yield of 2  10 -4 in water at 293 K—compared with about 0.8 in the protein. The mechanism of radiationless decay has been es- tablished as an internal conversion, promoted by motion along a volume conserving and nearly barrierless coordinate. 18,19 Efficient radiationless decay is observed in the neutral, anionic and cationic charge states of HBDI, with slightly different rates. 18,19 The nature of the coordinate leading to radiationless decay has been the subject of a number of quite detailed theoretical calcu- lations. 20-30 Single bond rotations about both bridging bonds have been considered. 20,23,24 Under different sets of conditions, Figure 1. Structures of the alkyl derivatives of HBDI studied in this work. Received: December 6, 2010 Revised: January 5, 2011