Fluorescent tags have become a staple of biological experiments since their prototype — the green fluorescent protein (GFP) of the jellyfish Aequorea victoria — was cloned in 1992. The protein glows green when it is excited by blue light, the result of the transfer of a proton between two amino acids. Richard Mathies and his colleagues at the University of California, Berkeley, have now teased apart the changes in GFP's atomic structure that allow this proton transfer to occur.

Mathies, a chemist, is interested in reaction dynamics, especially those involving photoexcitation. He has long been trying to visualize the structural changes that molecules undergo during chemical reactions as result of atomic motions that occur in the time range from tens of femtoseconds (10−15 seconds) to a picosecond (10−12 seconds). “This was no-man's-land for structural techniques,” he says. In 1997, during a sabbatical at the University of Leiden in the Netherlands, he realized that he could adapt Raman spectroscopy — a laser-based technique that detects the frequencies of atomic vibrations in molecules — to collect high-resolution vibrational spectra on the femtosecond timescale. “I was so excited I sent faxes to the lab in the middle of the night trying to get my students fired up and working on it,” he says.

Mathies first applied the technique, dubbed femtosecond-stimulated Raman spectroscopy, to the photoreaction of the visual pigment rhodopsin. During this process — which normally occurs in the retina of the eye — rhodopsin's excited state lasts for only 50 femtoseconds, too short a time for the researchers to monitor the evolution of the excited state, even with the new technique. “Nevertheless, we gained a lot of information about the first step in the visual process,” says Mathies. Then graduate student Renee Frontiera suggested trying the technique with GFP, which remains in its excited state for 5–10 picoseconds.

A talented new postdoc, Chong Fang, took up the project and developed a system of multiple lasers with the appropriate wavelength ranges — a process that took several years. The group got their first taste of success when they used the system “to obtain really good signal-to-noise vibrational spectra of GFP every 25 femtoseconds,” says Mathies.

By analysing hundreds of these spectra, they reconstructed the structural alterations that occur in the excited GFP molecule as it undergoes proton exchange (see page 200). “If you are trying to drive from one place to another, the simplest way, in theory, is to follow a straight line from A to B,” says Mathies. “But, as we know from driving in any city, the reality is not that simple — you might first have to go through a tunnel, then up a ramp and then onto the freeway. The same thing happens with GFP. The proton is not simply handed from one amino acid to another. The molecule undergoes a series of skeletal changes that eventually align the atoms in just the right way for proton transfer.”

Although the findings provide a detailed description of excited-state proton transfer — a widespread chemical reaction — the work paves the way for even more detailed studies. “The lasers used in this study were put together years ago, limiting us to 25–50 femtosecond resolution,” Mathies says. “With up-to-date lasers, we could get down to 10 femtoseconds. That would be unprecedented.”