Vibrational Spectroscopy 62 (2012) 1–6 Contents lists available at SciVerse ScienceDirect Vibrational Spectroscopy j ourna l h o me page: www.elsevier.com/locate/vibspec Short communication Ultrafast vibrational dynamics of parallel excited state proton transfer reactions in the Green Fluorescent Protein Jasper J. van Thor a,∗ , Craig N. Lincoln a , Brendon Kellner b , Konstantinos N. Bourdakos a , Lee M. Thompson c , Michael J. Bearpark c , Paul M. Champion b , J. Timothy Sage b,∗∗ a Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom b Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, MA 02115, United States c Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom a r t i c l e i n f o Article history: Received 12 September 2011 Received in revised form 13 March 2012 Accepted 15 March 2012 Available online 23 March 2012 Keywords: Green Fluorescent Protein Excited state proton transfer Photoselection measurements Ultrafast infrared spectroscopy a b s t r a c t We present a brief review of the current understanding and analysis of the photocycle of the Green Flu- orescent Protein (GFP). GFP is unique to show directed excited state proton transfer (ESPT) in a protein environment, which provides a directional coordinate for the ultrafast proton transfer reactions in con- trast with disordered liquids. ESPT proceeds on a picosecond time scale and we consider details of the vibrational response of the chromophore and the protein environment during the course of this reac- tion. In addition we discuss both experimental and computational methodology and corrections that measure and model vibrational dichroism from polarised pump-probe infrared measurements. For the GFP photocycle, a direct relationship between equilibrium protein side-chain conformation of glutamate 222 and reaction kinetics has been established for the ultrafast ESPT in the fluorescence photocycle. We have resolved the infrared spectral differences between heterogeneous ESPT reaction dynamics that were assigned to the carboxylate of the Glutamate 222 side chain. We additionally discuss photoselection mea- surements for the molecular interpretation of the vibrational transition dipole moments placed in the X-ray frame as a sensitive probe of the mode character and assess the assignments based on frequency calculations from the analytical second derivative for the isolated chromophore. Dipole gradients can be calculated analytically, or numerically by finite difference. An older software release that displays analytical dipole gradients incorrectly is identified. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Green Fluorescent Protein (GFP) from Aequorea victoria is a widely used marker protein for fluorescence bioimaging tech- niques in vivo [1]. The photochemical characteristics of GFP arise from highly specific protein–chromophore interactions and are very different from the isolated p-hydroxybenzylidene- imidazolinone (HBDI) chromophore in the condensed state, where radiationless decay, associated with rapid twisting motions in the excited state [2,3], is dominant. In GFP the specific protein environ- ment inhibits these and other rapid deactivation reactions [2–4] and in addition provides specific H-bonding interactions with the HBDI chromophore that are functional in the light-driven reactions Abbreviations: GFP, Green Fluorescent Protein; ESPT, excited state proton trans- fer. ∗ Corresponding author. Tel.: +44 20 75945071. ∗∗ Corresponding author. Tel.: +1 617 373 2908. E-mail addresses: j.vanthor@imperial.ac.uk (J.J. van Thor), jtsage@neu.edu (J.T. Sage). of GFP (Fig. 1). In the ground state, GFP exists predominantly in the neutral phenol state, with the phenolic p-hydroxybenzylidene- imidazolidinone chromophore protonated. The dominant but partial stabilisation of this species, GFP A ( max = 398 nm) at pH 8.0 results from electrostatic repulsion by an acidic group, identified from X-ray crystallography to be the carboxylate of Glu222 [5,6]. The thermal equilibrium properties include a plateau in the pH titration in this region that is predicted from a four-state system that describes the interaction of the two ionisable groups [7]. It has been known since the early purification and characterisation of native GFP from jellyfish that fluorescence emission with blue excitation is green, and hence that the effective Stokes shift for the GFP A state is very large, in contrast to that of the GFP B state, which is the minor population with anionic chromophore in the ground state absorbing maximally at 475 nm [8–10]. The size of the very large Stokes shift, as an energy term, is taken as an approximation for the loss of proton affinity in the excited state, minus relaxation in the Franck Condon regions [11,12]. The resulting drop of the pK a of the chromophore with optical excitation drives the ultrafast, picosecond time-scale, ESPT reaction. For the ESPT reactions a sig- nificant H/D kinetic isotope effect has been demonstrated at room 0924-2031/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2012.03.005