Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy
Citation: Chong Fang, Renee R. Frontiera, Rosalie Tran & Richard A. Mathies (2009/11/12) Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy. Nature 462, 200-204 (RSS)
DOI (original publisher): 10.1038/nature08527
Semantic Scholar (metadata): 10.1038/nature08527
Sci-Hub (fulltext): 10.1038/nature08527
Internet Archive Scholar (search for fulltext): Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy
Download: http://www.nature.com/nature/journal/v462/n7270/pdf/nature08527.pdf
Tagged: Chemistry
(RSS) PhotoChemistry (RSS)
Summary
For complex chemical and biological transformations, mapping out the reactive potential-energy surfaces describing atomic motions and resultant reaction dynamics has always been challenging.It has been possible only for simple reactions. This paper is based on tracing the transient atomic motions that lie at the heart of chemical reactions.Skeletal motions involved in the proton transfer producing fluorescent form of the protein has been studied using femtosecond stimulated Raman Spectroscopy (FSRS) techniques. In particular, it was observed 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. These oscillations were further attributed to impulsively excited low-frequency phenoxyl-ring motions, which optimize the geometry of the chromophore for ESPT.
Basically this vibrational structural technique uses a femtosecond actinic pump pulse to initiate photochemistry, and a picosecond Raman pump pulse together with a 20-fs Raman probe pulse to stimulate broadband Raman signals within a possible vibrational range of 50–3,500 cm-1. The time resolution of FSRS is determined by the cross-correlation between the femtosecond actinic pump and Raman probe pulses.The energy resolution is determined by the Raman pump bandwidth and the vibrational-coherence free-induction decay time.
METHOD SUMMARY:
The wild-type GFP sample was expressed from plasmids containing the His-tagged wild-type GFP gene and prepared in both H2O and D2O buffer solutions at ~pH 8 following standard methods. Output of a 1-kHz Ti:sapphire regenerative amplifier was used to generate the 80-fs, 400-nJ actinic pump pulse at a wavelength of 397 nm 20-fs, 7-nJ Raman probe pulse was obtained at 840–960 nm by continuum generation and prism compression. 3-ps, 2-μJ Raman pump pulse at 794 nm was produced by spectrally filtering ~90% of the amplifier output. At the focal point of the three pulses, 600 μl of A396 nm = 1 sample solution was flowed through a 1-mm cell. The probe pulse was disperserd which was carrying the stimulated Raman gain features in a spectrograph and recorded it using a dual-photodiode array, from which the time-resolved excited-state spectra were generated, processed and analysed.
The 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.
