Isomerization in Fluorescent Protein Chromophores Involves Addition/ Elimination Jian Dong, Fardokht Abulwerdi, † Anthony Baldridge, Janusz Kowalik, Kyril M. Solntsev, and Laren M. Tolbert* School of Chemistry and Biochemistry, 901 Atlantic DriVe; Georgia Institute of Technology, Atlanta, Georgia 30332-0400 Received May 7, 2008; E-mail: laren.tolbert@chemistry.gatech.edu Fluorescent proteins related to the green fluorescent protein (GFP) are thought to undergo Z/E photoisomerization between fluorescent and dark states. The Z form (“cis”) is the resting fluorescent form, while the E (“trans”) form is nonfluorescent, although exceptions are known. 1 Such proteins are also characterized by “blinking”, that is, temporary conversion to a nonfluorescent form, which has been variously attributed to triplet formation 2 or proton transfer. 3 Additionally, strong support for cis/trans isomerization is provided by the behavior of kindling fluorescent proteins, 4 in which the resting nonfluorescent form has trans stereochemistry. Upon irradiation into the long-wavelength band, the protein is “kindled” to the fluorescent cis form. That kindling is the result of photoisomerization is given strong support by recent single crystal X-ray determinations of both forms of two kindling proteins, dronpa and mTFP0.7, which differ in the stereochemistry about the benzylidene bond of the chromophores. 5 A key unresolved issue in the photophysics of the fluorescent proteins is whether the cis/trans isomerization is related to the blinking phenomenon, which, in addition to isomerization, has been ascribed to protonation and triplet formation mentioned earlier. We have observed that, in solution, the isolated UV-excited GFP chromophore undergoes a fast relaxation to yield a resting state which shows considerable twisting 6 at the same time that the excited-state is quenched. 7 In the protein, however, the common view is that the protein prohibits twisting about the double bond. Nonetheless, both calculations 8 and the aforementioned kindling behavior require that, in at least some instances, formal isomerization, that is, decay from the twisted intermediate onto the trans hypersurface, must be permitted. Moreover, the quite wide variation in blinking behavior as a function of protein structuresconditions which either facilitate or inhibit such isomerizationssuggest that blinking and isomerization are intimately involved. While the photoisomerization mechanism has been the subject of several studies 9 and corresponds in unexceptional ways to the mechanisms of other arylidene chromophores, the mechanism of the thermal reverse isomerization is more problematic. The blinking phenomenon requires that isomerization, if involved, be thermally reversible. Tonge has recently measured the rates of thermal isomerization of the representative E protein chromophore p- hydroxybenzylidenedimethylimidazolinone (HOBDI,X ) OH) 10 following photoisomerization from the Z form and obtained a barrier of 13.1 kcal/mol for the isomerization from an Arrhenius plot. 11 Surprisingly, little account has been taken of the observation that a high level ab initio calculation from Weber, et al., produces a barrier of 57 kcal/mol, 12 a value more typical of double-bond isomerization barriers for unexceptional benzylidene molecules such as HOBDI. Alternatively, tautomerization to a zwitterionic inter- mediate via an uncalculated transition structure, which then rotates with a 7.3 kcal/mol 12 barrier, presents another facile mechanism for isomerization. This poses a conundrum: how does one resolve the discrepancies between highly credible experimental and theo- retical determinations? To develop further insight into this process, and to exclude possible proton-transfer processes in the isomerizations, we first examined the methyl ether of HOBDI, MeOBDI. In the process of exploring the optimal solvents for our physical studies, we encountered a surprise. That is, in benzene and acetonitrile, no thermal isomerization occurred, but isomerization was readily observed in methanol and, at slower rates, in Me 2 SO-d 6 . 13 Indeed, we were able to isolate the trans isomer of MeOBDI by silica gel chromatography and record its NMR, ir, and electronic absorption spectra (see Figure 1 and Supporting Information). 14 Again, this result is inconsistent with a facile thermal isomerization, and suggests that a more complex process is intervening. These waters were further muddied by a recent claim 15 that an analogous 4-methylbenzylidene derivative does not isomerize under † REU student, Department of Chemistry and Biochemistry, University of Tulsa, Tulsa, OK 74104. Scheme 1. Isomerization in the GFP Chromophores Figure 1. UV-vis (top) and IR (bottom) absorption spectra of E and Z isomers of p-MeOBDI in MeCN. Published on Web 10/01/2008 10.1021/ja803416h CCC: $40.75  2008 American Chemical Society 14096 9 J. AM. CHEM. SOC. 2008, 130, 14096–14098