Playing protein tag: Repeatable NMR

Watching protein behaviour unfold

 

Chemists at the University of California San Diego have developed a method that allows them, for the first time, to attach chemical probes on to proteins and remove them again in a straightforward and repeatable cycle. The method will facilitate NMR spectroscopy for metabolic and other research studies.

Michael Burkart and his colleagues have spent a decade developing a technology that allows them to tag a protein with a removable chemical probe at a specific point in the macromolecule. Their approach means that they can attach a wide variety of probes as well as endowing the technology with many different potential applications. The work will ultimately offer new insights into how proteins function in order to direct research into better antibiotics, novel anticancer drugs, biofuels and other products. It will facilitate biomedical and biotech research directly allowing proteins to be purified more easily and tracked in living cells.

For instance, Burkart's laboratory hopes to understand the biochemical pathways involved in the metabolism of fatty acids as well as the biosynthesis of natural products. They have engineered algae to produce an improved biofuel profile of oils. They hope that new insights will allow them to optimise this process still further and to augment or even displace existing fossil fuels.

"In fatty acid metabolism, the fatty acids grow from an arm that eventually curls around and starts interacting with the metabolic protein," explains Burkart. "What we wanted to know was how long does the growing fatty acid get before it starts binding with the protein?"

To help them in this endeavour, Burkart and colleagues Nicolas Kosa, Robert Haushalter and Andrew Smith developed a way to remove the chemical probe from a metabolic protein using the microbial enzyme phosphodiesterase derived. They could reattach a fatty acid analogue to refold the protein and could repeat the process many times, all the while tracking the changes with NMR spectroscopy at the different metabolic stages. They could thus follow the biochemical pathway of the fatty acid metabolism.

"Without this tool, we would really have very limited ways of studying the dynamics of these fundamental metabolic processes," Burkart adds. "This opened the door for us to finally examine in detail the fatty acid biosynthesis shared by algae, which you have to understand if you want to engineer ways to improve the quantity of oil that's made by algae or to make different types of oil molecules in algae that are better for biofuels."

The team also used NMR to verify that the process of chemically removing and attaching the chemical probes was not itself altering the protein. "We've shown that we can do this iteratively, at least four or five times, without any degradation of the protein," says Burkart. "The protein remains very stable and can be studied very easily."

Many other biochemical processes exploit similar metabolic pathways on the way to many natural products used in pharmacology, agrochemicals and elsewhere. Burkart says their new tool could be used widely in chemistry laboratories across the globe.

Tag attachment

"One could attach a tag, such as biotin, that would allow the protein to be purified. Then one can clip off the tag and attach a fluorescent molecule to monitor protein interaction with other molecular partners," explains Burkart. "The method could also be used for studying living cells, such as observing protein expression levels throughout the cellular life cycle. We certainly see that as a possible application."