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It took Schultz and his team approximately 5 years and many
dead ends to solve the problem of generating an orthogonal
tRNA/synthetase pair. They then tackled the job of altering
the synthetase/amino acid specificity using a number of different
in vitro evolution strategies. Recently they hit on
a general method which after a few rounds of molecular evolution
allowed Wang to generate a synthetase that loaded an orthogonal
tRNA with 99.99 percent fidelity. "It worked surprisingly
well," says Schultz.
"You just need to add the novel amino acid to the culture
and grow the cells," says Wang.
Using this method, they incorporated O-methyl-L-tyrosine
into proteins with fidelity greater than 99 percent, which
is close to the translation fidelity of natural amino acids.
"We really wanted high fidelity, and we thought if we could
get it with O-methyl-tyrosine or tyrosine or phenylalanine,
then one could probably get high fidelity with [almost] any
amino acid," says Schultz.
Some wondered whether the feat could be repeated. One of
the reviewers of the paper told Schultz that the result was
amazing but a dead end. "He said O-methyl worked because it
is very similar to tyrosine and we would never be able to
do it again ," says Santoro.
"The next day," says Schultz, "Lei came in and he had just
added [a] napthyl alanine to the code." Since that day, Schultz's
group has added a host of novel amino acids to the E. coli
genetic code.
Novel X and Four-Base Codons
There are novel amino acids that contain fluorescent moieties
that are smaller than green fluorescent protein. These can
be used in place of GFP to label proteins and observe them
in vivo. Others novel amino acids useful as molecular
probes have side chains that can be phosphorylated or that
contain spin labels.
There are novel hydrophobic amino acids, which should be
useful for probing structure, and novel nucleophiles, heavy
metal-binding amino acids, and photoisomerizable side chains,
all of which should confer new activity to the proteins. There
are novel glycosylated amino acids that could be used to make
therapeutic proteins, and there are novel amino acids with
keto groups that can be used to selectively label proteins
with practically any molecular group of interest.
There are also groups that contain photoaffinity labels
that could be used for covalently cross-linking proteins to
one another in a photoinduction proteomics experiment.
"The idea," says Postdoctoral Fellow Jason Chin, "is that
you will put photo-crosslinking groups into a specific site
in a protein. You could then see what the protein interacts
with in living cells. And you will be able to look at weak
interactions that are difficult to detect by current methods."
Putting in many different novel amino acids is the next
step, and Schultz is working on generalizing the method used
for the O-methyl-tyrosine so that he can routinely do this.
Schultz believes the key to easily inserting a new amino
acid is changing the specificity of the aminoacyl-tRNA synthetase.
Schultz describes the process as "gutting" the active site—putting
in a hole that can be filled with a combination of a new amino
acid and a new protein side chain. However, not all pegs fit
in the same hole, and some synthetases will not be able to
take certain novel amino acids. But by making several of these
tRNA/synthetase pairs, it should be possible to put in almost
any amino acid.
One pair that is underway is the leucine synthetase pair,
which Schultz and his graduate student Christopher Anderson
are working on now. This pair is interesting because it may
be used to expand the technology beyond the amber codon, which,
though successful and robust, is limited. "That only lets
you use one or potentially two [different] amino acids [per
protein]," says Schultz.
"We're developing the leucine synthetase system to recognize
a four-base codon," says Anderson.
The difficulty, though, is that most anticodon loops are
key recognition elements of the synthetase, and this recognition
becomes intolerably perturbed due to the structure of the
four-base tRNAs. "You have to be able to change the anticodon
loop of the tRNA and still be able to have the synthetase
recognize the tRNA," says Anderson. In the leucine synthetase
system this is not a problem because the synthetase recognition
occurs at another site.
The strategy involves using molecular evolution experiments
to select for tRNAs with anticodon loops that recognize four
or five bases. The advantage of using the longer codon is
diversity—there are 256 four-base codons possible, for
instance, and many of these can be re-assigned to a new unnatural
amino acid. They are now building new tRNA/synthetase pairs
that decode four bases at a time.
Life with Many Amino Acids
Another interesting question the group is working out is
how to transfer the technology to eukaryotic cells. "Right
now all this work has been done in E. coli," says Santoro.
"It would be much more interesting to be able to express proteins
containing unnatural amino acids in mammalian cells."
" The ability to do unnatural cell biology by introducing
unnatural amino acids such as flurophores and photocrosslinkers
into proteins in eukaryotic cells will provide a powerful
arsenal of tools to dissect and understand how these cells
and even whole organisms work," says Chin, who is working
on expanding the eukaryotic code. "We will be able to probe
protein interactions involved in human disease with unprecendented
precision in living cells."
Another follow up project applies the technology in a random
way, adding novel amino acids to the genetic code of cells,
allowing the cells to use them. The team will see how these
changes affect the organism's ability to, say, evolve in response
to stress.
To answer this, Schultz and his colleagues plan to do a
random unnatural amino acid mutagenesis of the entire E.
coli genome and subject the new cells to some sort of
selective pressures—like heat or antibiotics—and
see what happens. Could a 21-amino-acid bug be more robust
than a 20-amino-acid bug?
"If you put that cell under some stress, will the cell be
able to evolve faster to deal with that stress?" asks Schultz.
A further application of this line of research that Schultz
is contemplating is to randomly mutate proteins with several
unnatural amino acids in many places simultaneously. And yet
another possibility would be to add the metabolic pathway
for the synthesis of the novel amino acid to the cells so
that it would not have to be added to the growth medium—something
that Wang, Anderson, and Zhang are addressing at the moment.
Polyester Proteins
Schultz and his team are also asking whether we need amino
acids in the first place. Can you use some other polymer building
block, like hydroxy acids, as a protein constituent and will
the resulting protein fold?
Whereas novel amino acids differ in side chain chemistry
but have the same amide backbone as natural amino acids, hydroxy
acids would have a completely different backbone structure.
Instead of a polyamide, they would form polyesters.
One of the first things they will try is simply to get a
single hydroxy acid incorporated site specifically, using
the same technology they worked out for the O-methyl-tyrosine
system. Then with this proof of principle out of the way,
they will proceed to trying to use two or more tRNA/synthetase
pairs that incorporate hydroxy acids that are based on hydrophobic
and hydrophilic amino acids in the hope of making a folded
polyester protein.
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