Oxford scientists unleash radical chemistry to explore novel protein activities through synthetic post-translational modifications.
Pioneering experiments carried out in the 1960s demonstrated that the reactivity of certain amino acid side chains could be exploited to alter the identity of an amino acid—for example, to transform a serine into a cysteine—or to enable the attachment of chemical groups that could then be used for downstream applications such as affinity labeling. Predating the rise of molecular biology and the ability to re-code proteins through genetic means, the approach was then termed chemical mutagenesis. Benjamin Davis of the University of Oxford says these “farsighted experiments” are what motivated work recently published by his group in Science, which describes how they succeeded in carrying out carbon-bond-forming reactions under biocompatible conditions, a method they named post-translational mutagenesis.
There are plenty of ways to modify amino acid side chains to add chemical groups, but lack of specificity is a pitfall, says Davis. “Because most side chains in proteins are made of carbon–carbon, you really want to make a carbon sp3-to-sp3 bond, and that's been a project that we've been toying with for quite a while,” he says; “the nice thing is if you make the carbon–carbon bonds close enough to the backbone, in principle you are kind of unlimited in terms of what you can slot in.”
Indeed, Davis and his colleagues demonstrate how radical chemistry can be used to efficiently add a wide array of natural or synthetic post-translational modifications in a site-specific manner through a two-step 'tag and modify' approach. The first step is to install a precursor for the reaction—the unnatural amino acid dehydroalanine (Dha)—at the desired position in the protein. This can be done through genetic, biosynthetic or chemical methods. Dha can then be converted into almost anything, in principle, through reaction of its double bond—from the first carbon to the carbon of the backbone—with selective radicals. The idea had been bouncing around in the lab for a while; the difficult part was identifying a reaction compatible with proteins. “We knew that we could make a carbon–carbon bond using zinc as metal, but it was messy,” Davis recalls.
It was by studying the underlying mechanism of those zinc-containing reactions that graduate student Tom Wright, the first author of the paper, led the team to the realization that zinc was not necessary. Instead, radical precursors could be used to efficiently generate the desired bond under biocompatible conditions. “It was a chemist's mindset—to try to pick the chemical mechanism—that allowed us to make something that was more efficient in the context of biochemistry,” says Davis.
The group went on to demonstrate the versatility of their method by installing a variety of modifications on proteins, including glycosylations for which no convenient general chemical method was available. Going the extra mile, they also tested the behaviors of the modified proteins in a variety of biochemical assays. The pilot implementations of the method, along with follow-up experiments, have already yielded interesting insights in glycobiology and epigenetics.
A potential limitation, however, is that both D - and L-configurations are present at the modified site. Despite this, Davis says that even with a mixture, the method already enables researchers to gain significant biochemical insights. However, he is quick to add that enantiomer specificity is desirable. “It would be lovely, as a next stage, to move towards that, and that's our next goal,” Davis confides. In the meantime, the group hopes that this chemistry will provide a useful addition to the synthetic biologist's toolbox.
Wright, T.H. et al. Posttranslational mutagenesis: a chemical strategy for exploring protein side-chain diversity. Science http://dx.doi.org/10.1126/science.aag1465 (2016).
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Larochelle, S. Extreme makeover: protein edition. Nat Methods 13, 907 (2016). https://doi.org/10.1038/nmeth.4063