Non-Condon Effects in the One- and Two-Photon Absorption Spectra of the Green Fluorescent Protein

We report calculations of one and two photon absorption (OPA and TPA, respectively) spectra of the green fluorescent protein (GFP) chromophore using the doubleharmonic parallel-mode approximation and including explicit dependence of the electronic transition moments on nuclear geometry. The non-Condon effects are found to be more significant for TPA resulting in a different shape of the spectra and a blue shift of 500 cm of the TPA peak absorption relative to OPA. The computed shift is in excellent agreement with the experimentally observed 700 cm. SECTION: Biophysical Chemistry T green fluorescent protein (GFP) has revolutionized the fields of cell and molecular biology by permitting specifically targeted cellular imaging. Recently, there has been considerable interest in using GFP in conjunction with two-photon laser scanning microscopy (TPLSM). The longer wavelengths used in fluorescence imaging via two-photon excitation offer the advantages of greater penetration depth, reduced photodamage and photobleaching, and smaller background fluorescence. Two-photon absorption (TPA) experiments on GFP, however, have revealed a systematic blue shift of the absorption peak from one-photon absorption (OPA) experiments for which the cause is not fully understood. Since there is also significant interest in the preparation of “designer” fluorescent proteins, which are capable of absorbing and emitting at specific wavelengths, an understanding of the factors that affect the TPA spectrum is important. Several explanations for the observed 500-750 cm shift of the absorption peak have been posited. One possible explanation is the presence of a nearby excited state that is dark under OPA, but bright for TPA. Previous calculations in our group, however, show no evidence of a dark state in close proximity to the bright πfπ* state. Moreover, the presence of a dark state within less than 1000 cm from the bright state would result in strong vibronic couplings, intensity borrowing, and reduction in the fluorescence yield, which is not observed experimentally. Finally, as pointed out in refs 8 and 10, it is unlikely that such a dark state is present within the same energy interval from the bright state in many of fluorescent proteins (see, for example, ref 10) and in other molecules for which blue-shifted TPA spectra have been reported. An alternative explanation, which we pursue in this letter, is that the change in the transition moment along some normal coordinates leads to preferential transition into excited vibrational levels of the S1 state. The transition of interest is πfπ*, and a Huckel-like model predicts that the transition dipole moment depends sensitively on the geometry of the bridge moiety (see eq A4 in ref 16). Since a number of the Franck-Condon active modes involve stretching or bending around the central carbon atom of the chromophore, one can anticipate significant nonCondon effects for the absorption spectra. The vibronic effects in TPA absorption have been noted before and suggested as a possible explanation of the blue-shifted TPA in fluorescent proteins, however, no first-principle calculations have been performed to support this hypothesis. This letter is organized as follows. In the next section we describe the details of how we compute the absorption spectra, including how we obtain geometries and OPA and TPA cross sections. The following section compares our calculated OPA spectra with gas-phase experiments on the model chromophore and with wild-type GFP in the native protein environment. We also compare our calculated OPA and TPA spectra. Finally we Received: November 30, 2010 Accepted: January 30, 2011