Green glow deciphered
Mysterious jellyfish gene widely used in biology find its place in nature.
A protein that glows green, and won a Nobel prize for its discoverers and developers, has finally found its role in life — to paint the world red.
Since it was isolated from jellyfish in the early 1960s, green fluorescent protein (GFP) has been used as a biological tool to track other proteins within cells. The GFP gene is attached to the gene for the protein of interest and inserted into cells. The cell then produces both proteins together, allowing the target protein to be monitored by the green glow GFP gives off.
But GFP's natural function has remained a mystery — as well as jellyfish, it is naturally present in a wide range of animals and plants, including some species of coral and fish.
Now it seems that the green protein can donate electrons in a process powered by light, to molecules that like to accept electrons. This is what Konstantin Lukyanov of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry in Moscow, Russia, and his colleagues found out by accident1. Such electron donors play key roles in a variety of cellular processes and could, for instance, mean that GFP is used to sense light in some organisms.
Lukyanov's team was looking at another poorly-understood property of GFP — starve the protein of oxygen, and the green glow turns red. Whilst looking at the effect of external molecules on this process he set up control groups that were exposed to oxygen.
The researchers expected to see nothing in the control groups. But instead, Lukyanov noticed that in the presence of molecules that could accept electrons, such as benzoquinone or potassium ferricyanide, when light hit the system there was 'redding' — the same response as seen in low-oxygen conditions. The team hypothesised that when GFP is exposed to light, excited electrons hop from it to electron-accepting molecules nearby. That could cause changes to the structure of GFP's chromophore — the part of GFP responsible for its colour — perhaps accounting for the colour change.
They next looked at what happened in the presence of biologically relevant molecules, such as the protein cytochrome c and nicotinamide adenine dinucleotide, both of which help cells to extract energy from sugars, are also capable of accepting electrons. Once more the light-excited GFP turned red, suggesting that it was again donating electrons to these molecules.
"It is quite unexpected that GFP can interact with external molecules and donate electrons," says Lukyanov, because its chromophore is locked away inside a protein shell.
The team then looked at GFPs from a wide range of sources, including different jellyfish and plankton species, in the presence of benzoquinone and potassium ferricyanide. All organisms experienced redding, apart from mutant versions of GFP that glow blue and cyan.
The researchers also introduced the GFP gene into live mammalian cells grown in the lab, and found the redding process happened there too. This suggests that scientists could use GFP to detect reduction and oxidation processes inside a cell, Lukyanov says.
"We found redding to occur in all tested GFPs taken from diverse animals," says Lukyanov, suggesting the process, which is powered by light, may have evolved many years ago. "It's similar to photosynthesis," he adds.
The team still doesn't know exactly what is happening during redding, but they managed to look in more detail at cytochrome c, and they think it involves the transfer of two electrons. Two cyctochrome c molecules are reduced, or given an electron, for each GFP molecule that gets excited by light. They report their results in Nature Chemical Biology.
Marc Zimmer, a computational chemist at Connecticut College in New London who works with GFP, thinks that Lukyanov has the best evidence yet for what GFP does in nature, and is impressed that the effect is so widespread. "It presents an answer to a puzzle that we've had for a while," he says.
However, the case is not closed: "What about all the other coloured fluorescent proteins?" asks Zimmer.
Lukyanov says that the latest work is a hypothesis, but he thinks that other biologists who use GFP as a tag should bear it in mind, because it might affect their experiments. "It might be especially important in cases when GFP is fused to redox-active partners, for example cytochromes or flavoproteins," he says.
- Bogdanov, A. M. et al. Nature Chem. Biol. doi:10.1038/nchembio.174 (2009).