Novel Amino Acids Come of Age

By Jason Socrates Bardi

Some time in the few hundred million years between the formation of earth's crust and the emergence of earth's oldest known cells, cyanobacteria, protein synthesis evolved. Whether it evolved in cyanobacteria 3.5 billion years ago or in some earlier precursor decades before is unknown. But we do know that the proteins were synthesized using only 20 amino acids because all higher organisms that have come after have used the same 20.

"Why do all forms of life we know use the same common 20 amino acids and only 20 (seleno cysteine excluded)?" asks Peter Schultz, professor of chemistry and Scripps Family Chair of The Skaggs Institute of Chemical Biology. "Can we change this by adding new amino acids to the genetic code and can we use these new amino acids to change the structures and functions of proteins in interesting ways?"

The answer to his last three questions is now, apparently, yes.

About 15 years ago, when Schultz was at the University of California at Berkeley, he began inserting novel amino acids into proteins in vitro, and about five years ago, he began working on a project to do it in vivo. "We are interested in proteins because, as chemists, the most fascinating class of molecules are proteins, which have functions ranging from photosynthesis to signal transduction to gene regulation." says Schultz.

"And," he adds, "when chemists look at molecules, they say, 'How can we better understand how they work, and how can we rationally manipulate their structure to create interesting new functions?'"

Schultz reckoned that it would be possible to enhance the chemical, physical, and biological properties of proteins by adding novel amino acids—ones that are not among the 20 that all living organisms use. He wanted a way to do this easily and in vivo, because direct chemical or biochemical synthesis of a protein containing unusual amino acids, while possible, is limited, laborious, and low-yielding.

"To make proteins in a robust way, one has to do it inside cells—it's difficult to synthesize proteins in a test tube," says Schultz.

Success Story

Schultz, his student Lei Wang, and their colleagues announced in a Science paper earlier this year that they had succeeded in adding the new amino acid O-methyl-tyrosine to the genetic code of E. coli. O-methyl-tyrosine is the first of several new amino acids they are working on site-specifically inserting in vivo—a proof of principle.

Also called "unnatural" because they are not among nature's original 20, these novel amino acids have the same carboxy–amino backbone as the 20 standard amino acids but different side chains. Some have just slightly altered chemical structures and others have new functional groups added. In proteins, these differences may alter everything from structure and folding to activities. Certain "designer" side chains may even impart novel functionality.

With his new methodology, Schultz believes it will not be long before this technology is here. "It should be possible to put in a fluorescent probe or a photoaffinity label at any site on any protein in the cell," says Schultz.

The idiom, "You can't teach an old dog new tricks" doesn't even come close to describing how difficult it should have been to make organisms incorporate novel amino acids into their protein chains. After all, every organism in nature has been using the same 20 amino acids since the primordial soup. Organisms have to maintain fidelity in replication, and so life has evolved myriad mechanical ways of making sure only those 20 amino acids get incorporated into proteins.

Schultz and his colleagues did it anyway.

Working the Code

When a protein is expressed, an enzyme reads the DNA bases of a gene (A, G, C, and T), and transcribes them into RNA (A, G, C, and U). This so-called "messenger RNA" is translated by another protein–RNA complex, called the ribosome, into a protein. The ribosome requires the help of transfer RNA molecules (tRNA) that have been "loaded" with an amino acid. Protein specificity comes from the fact that the tRNA recognizes only one codon and gets loaded with only the one amino acid that is specific for that codon.

For every codon on the mRNA—every three bases—the ribosome attaches another amino acid to the chain. But even though there are 4x4x4 = 64 different codons (UAG, ACG, UTC, etc.) there are only 20 amino acids that all organisms use to produce proteins. Some of the 64 codons are redundant, with several coding for the same amino acid, and three of them are nonsense codons—they don't code for any amino acids.

These nonsense codons are useful because normally when a ribosome that is synthesizing a protein reaches a codon that does not code for any tRNA, the ribosome dissociates and synthesis stops. Hence nonsense codons are also referred to as "stop" codons. One of these, called the amber stop codon, UAG, played an important role in Schultz's research.

If the cell is provided with an amber suppressor—a tRNA that recognizes UAG—then the ribosome will grow the chain with the amino acid that this new tRNA carries and synthesis does not stop. Any codon in an mRNA that is switched to UAG will carry the new amino acid in that place and the ribosome will resume reading the full-length message.

Site-directed mutagenesis is an application of amber suppression, and by simple extension, a tRNA that recognizes UAG and carries a novel amino acid could be used to site-specifically incorporate novel amino acids. If he could create an amber suppressor tRNA that carried a novel amino acid and an enzyme to load the amino acid on that tRNA, Schultz knew he would have the ability to site specifically add the novel amino acid to a growing protein chain wherever he inserted the codon UAG.

All he needed was a tRNA unique to that codon and a synthetase "loading" enzyme to load the unnatural amino acid on that tRNA. Then he would have a simple and robust way to put a completely original residue anywhere he wanted in a protein.

99.99 Percent Fidelity

This was not easy. "It's hard to find synthetases that recognize novel amino acids because nature has designed them to recognize only the natural ones," says Research Associate Steve Santoro, a TSRI graduate. And overcoming that obstacle, there is also the requirement that the new tRNA/synthetase pair be functionally orthogonal, meaning the new pair must not interact with any of the existing tRNAs or synthetases.

"The new tRNA should not be aminoacylated by any existing synthetase, and likewise, the new synthetase should not recognize any existing tRNA," says Wang, who has been working on the project from its early beginnings. "There should be no crosstalk."

The orthogonal synthetase had to be engineered so that it charged the orthogonal tRNA with an unnatural amino acid but not any natural amino acids and do so with a fidelity that is as high as normal—99.9 percent or higher—something that proved difficult.

"That was the goal," says Schultz, "though we thought we wouldn't get there in ten years."


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"Historically, chemists have been interested in synthesizing molecules," says Professor Peter Schultz. "We are interested in synthesizing function." Photo by Biomedical Graphics.











The chemical structures of the amino acid tyrosine (top) and the novel amino acid O-methyl-tyrosine (bottom).















"You just need to add the novel amino acid to the culture and grow the cells," says graduate student Lei Wang. Photo by Jason S. Bardi.