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Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy


Tracing the transient atomic motions that lie at the heart of chemical reactions requires high-resolution multidimensional structural information on the timescale of molecular vibrations, which commonly range from 10 fs to 1 ps. For simple chemical systems, it has been possible to map out in considerable detail the reactive potential-energy surfaces describing atomic motions and resultant reaction dynamics1, but such studies remain challenging for complex chemical and biological transformations2. A case in point is the green fluorescent protein (GFP)3,4,5 from the jellyfish Aequorea victoria, which is a widely used gene expression marker owing to its efficient bioluminescence. This feature is known to arise from excited-state proton transfer (ESPT)6,7,8, yet the atomistic details of the process are still not fully understood. Here we show that femtosecond stimulated Raman spectroscopy9,10 provides sufficiently detailed and time-resolved vibrational spectra of the electronically excited chromophore of GFP to reveal skeletal motions involved in the proton transfer that produces the fluorescent form of the protein. In particular, we observe that the frequencies and intensities of two marker bands, the C–O and C = N stretching modes at opposite ends of the conjugated chromophore, oscillate out of phase with a period of 280 fs; we attribute these oscillations to impulsively excited low-frequency phenoxyl-ring motions, which optimize the geometry of the chromophore for ESPT. Our findings illustrate that femtosecond simulated Raman spectroscopy is a powerful approach to revealing the real-time nuclear dynamics that make up a multidimensional polyatomic reaction coordinate.

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Figure 1: Ultrafast structural response of the photoexcited wild-type GFP chromophore.
Figure 2: FSRS spectra of wild-type GFP in D 2 O from -100 fs to 1 ps following 396-nm excitation.
Figure 3: Time-dependent vibrational structural features of the excited wild-type GFP chromophore in D 2 O.
Figure 4: Deconvolved marker-band frequency oscillations reveal multidimensionality of the reaction coordinate of wild-type GFP.


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Plasmids were provided by R. Wachter. We thank M. Marletta for the use of his laboratory facilities to express and purify GFP samples. We thank J. Dasgupta for discussions. This work was supported by the Mathies Royalty Fund.

Author Contributions C.F., R.R.F. and R.A.M. designed the research. C.F. and R.R.F. performed the spectroscopic measurements and DFT calculations. C.F. analysed the data and simulated the spectra. R.T. expressed and purified the protein samples. C.F. and R.A.M. wrote the paper. All authors discussed and edited the manuscript.

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Correspondence to Richard A. Mathies.

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Fang, C., Frontiera, R., Tran, R. et al. Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy. Nature 462, 200–204 (2009).

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