Searching out the mechanisms of fluorescence

New laser sources, innovative microscope objectives, and advanced image processing techniques are all important for optical microscopy. But of course, without fluorophores, there is no fluorescence imaging.

Since discovery of the original fluorophore, green fluorescent protein (GFP, which won the 2008 Nobel Prize in Chemistry for Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien), a whole range of fluorescent proteins have been discovered and exploited for research, drug discovery, and preclinical and clinical applications. Despite its importance to biomedicine, however, the fundamental mechanism of fluorescence is still not well understood.

Modify and investigate

GFPs and comparable fluorescent molecules have similar structure, consisting of a "beta-barrel"—a hollow cylindrical protein structure—with a chromophore in the middle. There's a continuing effort to widen the library of available fluorescent proteins, and one method is to start from a robust, stable fluorescent protein, make minor modifications, and then investigate the properties of those modified proteins.

That's the general strategy used by Patricia Langan and her colleagues at Los Alamos National Laboratory. The group had previously developed a couple of extremely thermostable fluorescent proteins, eCGP123 and TGP. Using those as a starting point, they prepared "libraries" of proteins with one to three mutations at different places in each library. Then, they screened each library and used flow cytometry to select for the clones with the most shifted spectral properties. Those selected/shifted clones served as templates for the next round, where they introduced one to three new mutations, again selecting for various spectral properties. For each round, the proteins were produced in engineered E. coli. For this work, they selected specifically for photoswitchable proteins—those that can be turned on and off with light.

Photoswitchable, but not fluorescent

They ended up with a protein they named Dathail (after the Gaelic word for "colorful") that is interesting for several reasons. Although Dathail exhibits the beta-barrel/chromophore structure, it is not fluorescent. It has two "color" states of fairly well-defined absorbance: The ground state absorbs 389 nm light and transitions to a short-lived higher energy state that decays to a metastable state with an absorbance peak at 497 nm.

The chromophore in the ground state is in the cis-configuration, while the excited state generally transitions to the trans-conformation. In many fluorescent proteins, the cis-conformation is fluorescent, while the trans- is not. In Dathail, neither conformation is fluorescent. This wasn't the first indication that fluorescence required more than simply a chromophore in the cis-conformation, but it did provide Langan and her colleagues with the opportunity to investigate the mechanism further. The team performed both x-ray and neutron crystallography on the protein structure. Neutron crystallography is particularly interesting for fluorescent proteins, because proton-mediated energy transfer appears to be an essential factor in fluorescence.

For now, even without complete information on the mechanism, Dathail's photoswitchability and the spectral separation between the ground and excited state make it an excellent candidate for photochromic fluorescence (or Förster) resonance energy transfer (pcFRET) techniques. It also has promise as a tunable quencher to enhance the resolution of fluorescence microscopy.

In fluorescence imaging, as in most cases, technological advances and scientific understanding advance hand-in-hand.