Collapse and Recovery of Green Fluorescent Protein Chromophore Emission through Topological Effects

Abstract

Housed within the 11-stranded β-barrel of the green fluorescent protein (GFP) is the arylideneimidazolidinone (AMI) chromophore, the component responsible for fluorescence. This class of small-molecule chromophore has drawn significant attention for its remarkable photophysical and photochemical properties, both within the intact protein and after its denaturation. All of the proteins so far isolated that have visible light fluorescence have been found to contain an AMI chromophore. These proteins comprise an extensive rainbow, ranging from GFP, which contains the simplest chromophore, p-hydroxybenzylideneimidazolidinone (p-HOBDI), to proteins having molecules with longer conjugation lengths and a variety of intraprotein interactions. The fluorescence invariably almost vanishes upon removal of the protective β-barrel. The role of the barrel in hindering internal conversion has been the subject of numerous studies, especially in our laboratories and those of our collaborators. A better understanding of these chromophores has been facilitated by the development of numerous synthetic protocols. These syntheses, which commonly use the Erlenmeyer azlactone method, have evolved in recent years with the development of a [2 + 3] cycloaddition exploited in our laboratory. The synthetic AMI chromophores have allowed delineation of the complex photophysics of GFP and its derivatives. Upon denaturation, AMI chromophores are marked by 4 orders of magnitude of diminution in emission quantum yield (EQY). This result is attributed to internal conversion resulting from conformational freedom in the released chromophore, which is not allowed within the restrictive β-barrel. To date, the photophysical properties of the AMI chromophore remain elusive and have been attributed to a variety of mechanisms, including cis-trans isomerization, triplet formation, hula twisting, and proton transfer. Advanced studies involving gas-phase behavior, solvent effects, and protonation states have significantly increased our understanding of the chromophore photophysics, but a comprehensive picture is only slowly emerging. Most importantly, mechanisms in structurally defined chromophores may provide clues as to the origin of the "blinking" behavior of the fluorescent proteins themselves. One approach to examining the effect of conformational freedom on rapid internal conversion of the chromophores is to restrict the molecules, both through structural modifications and through adjustments of the supramolecular systems. We thus include here a discussion of studies involving the crystalline state, inclusion within natural protein-binding pockets, complexation with metal ions, and sequestration within synthetic cavities; all of this research affirms the role of restricting conformational freedom in partially restoring the EQY. Additionally, new photochemistry is observed within these restricted systems. Many of the studies carried out in our laboratories show promise for these molecules to be adapted as molecular probes, wherein inclusion turns on the f...

A three-state effective Hamiltonian for symmetric cationic diarylmethanes

“…44,60,72 Both these dyes and GFP chromophores display a common binding-dependent fluorescence enhancement associated with the suppression of a common double-bond photoisomerization chemistry in the excited state. [4][5][6][7][73][74][75] Our ongoing work shows that this analogy can also be applied to electronic structure along the photoisomerization coordinates of some of the dyes studied here. It does seem to us now that there is a general physics underlying the excited state behavior in all of these systems, and an opportunity for the development and application of quite general model Hamiltonians for fluorogenic monomethine dyes.…”

Fluorescent Proteins for Neuronal Imaging

“…Synthetic analogues of the FP chromophore have been extensively used for studying the effect of the chromophore environment on fluorescence emission [42,43]. one typical synthetic analogue is 4ʹ-hydroxybenzilidene-2,3-dimethylimidazolinone (hBdi; Fig.…”

“…The fluorescence of hdBi appears when the temperature is below the glass transition temperature of the solvent [44,46,48]. although there is still debate about the mechanism associated with this fast internal conversion, the current consensus attributes it to flexibility or rotation along one of the bonds adjacent to the methylene bridge [42,43]. The chromophore environment in FPs has evolved to prevent this internal conversion from occurring and thus lead to high fluorescence quantum yields.…”

Photophysics, Dynamics, and Energy Transfer in Rigid Mimics of GFP-based Systems

“…30−32 Therefore, the vast majority of chemically synthesized HBI-based analogs are exponentially (10 −4 ) less emissive in solution compared to GFP and its mutants. 31 Thus, to maintain emission of HBIbased chromophores, the β-barrel-chromophore interactions present in natural proteins must be replicated to restrict possible nonradiative decay pathways. Recent studies indicated the possibility to suppress the low-energy vibrational modes of HBI-based chromophores through their chemical modification.…”