The new technique allows scientists to add and remove different kinds of chemical probes at specific locations on proteins, such as the fatty acid molecule shown here. Image: J. LaClair, UC San Diego |
Chemists at the
University of California, San Diego (UC San Diego) have developed a method that,
for the first time, provides scientists the ability to attach chemical probes
onto proteins and subsequently remove them in a repeatable cycle.
Their
achievement, detailed in Nature Methods, will allow researchers to
better understand the biochemistry of naturally formed proteins in order to create
better antibiotics, anticancer drugs, biofuels, food crops, and other natural
products. It will also provide scientists with a new laboratory tool they can
use to purify and track proteins in living cells.
The development
was the culmination of a 10-year effort by researchers in the laboratory of
Michael Burkart, a professor of chemistry and biochemistry, to establish a
method to both attach a chemical probe at a specific location on a protein and
selectively remove it.
This flexibility
allows researchers to study the protein with many different functional
attachments, providing versatility akin to a biochemical Swiss Army knife. The
great advantage of this technique is the broad flexibility of the attachments,
which can be dyes, purification agents, or mimics of natural metabolic
products. Each of these attachments can be used for different purposes and
biological studies.
Burkart’s goal in
his own laboratory is to understand more about the biochemical pathways of
fatty acid metabolism and the biosynthesis of other natural products. One
project focuses on engineering algae in order to produce improved biofuels. In
this effort, the scientists hope to maximize the production of high quality
algae oils, which could be used to supplement or supplant 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,” says Burkart, who is also
associate director of the San Diego Center for Algae Biotechnology, or SD-CAB,
a consortium of institutions in the San Diego region working together to make
biofuels from algae commercially viable as transportation fuels. “What we
wanted to know was how long does the growing fatty acid get before it starts
binding with the protein?”
Burkart and
chemists in his laboratory—Nicolas Kosa, Robert Haushalter, and Andrew
Smith—found a way to remove the chemical probe from this metabolic protein
using an enzyme called a phosphodiesterase derived from the common bacterium Pseudomonas
aeruginosa. Subsequent reattachment of a fatty acid analogue reconstituted
the protein complex to its natural state. By repeating the process again and
again, while examining the molecular changes in the fatty acid with nuclear
magnetic spectroscopy, or NMR, during different metabolic stages, the
scientists were able to detail the biochemical pathway of the fatty acid
metabolism in a way they had never been able to do before.
“Without this
tool, we would really have very limited ways of studying the dynamics of these
fundamental metabolic processes,” Burkart says. “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 UC San Diego
chemists also used NMR to verify that the process of chemically removing and
attaching the chemical probes does not degrade or alter the protein in any way. “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.”
Because these
same metabolic processes are shared by the metabolism of many natural products,
including anti-cancer agents, antibiotics, and natural insecticides, Burkart
says this new tool should have wide application in natural product chemistry
laboratories.
“These are
fundamental biochemical pathways that we still don’t fully understand,” he says. “We’re now learning how these basic biosynthetic enzymes work. A large majority
of drugs are derived from natural products and many future medicines can result
from these pathways. There’s a great interest now in synthetic biology, using
these pathways to make new antibiotics or new anticancer drugs. They’re all
regulated by these same types of interactions.”
The UC San Diego
chemists say their method of tagging and removing chemical probes from proteins
should also have wide application as a general laboratory tool to visualize and
track proteins on living cells, as well as manipulate them outside of the cell.
