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 dynamics(1), but such studies remain challenging for complex chemical and biological transformations(2). A case in point is the green fluorescent protein (GFP)(3-5) 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)(6-8), yet the atomistic details of the process are still not fully understood. Here we show that femtosecond stimulated Raman spectroscopy(9,10) 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.