Proteins as Natural Products
By Jason Socrates Bardi
What chemists talk about when they talk about making molecules
is not necessarily what biologists talk about when they talk
about making molecules.
For many biologists, the problem is often one of finding
the right combination of expression system (E. coli
or yeast strain), DNA "vector," and purification scheme. The
best solution for a biologist is one that produces the most
copious amounts of highly purified molecules, be they proteins,
DNA, RNA, or carbohydrates, in the shortest amount of time.
For organic chemists, on the other hand, the problem of
making molecules is also one of finding the right combination
of steps. Natural products—those substances formed by
strange bacteria, tree bark, or other living creatures with
some useful property—are often chemists' targets. Making
these substances is not a problem of picking an expression
system, but of choosing among commercially available compounds
and designing a synthesis with the fewest, most elegant reactions.
The more complicated the architecture of the natural product,
the greater the challenge.
And how would someone who is both a chemist and a biologist,
say a bioorganic chemist, talk about making molecules? Would
it be synthesizing a natural product (like a chemist) or expressing
a protein (like a biologist)?
Both.
"The way that I think about proteins," says Assistant Professor
Philip Dawson, an investigator in The Scripps Research Institute's
(TSRI) Departments of Cell Biology and Chemistry and a member
of The Skaggs Institute for Chemical Biology, "is that they
are the ultimate natural products—molecules that we can
synthesize and, by having control over their chemistry, [design]
any side chain or backbone atom."
Dawson, who is a graduate of TSRI's Kellogg School of Science
and Technology, is working to move forward the methodology
of chemical protein synthesis. His goal is producing common
and novel protein forms, which are useful tools for studying
everything from the basic molecular basis of protein function
to applied questions of enzyme drug resistance. Currently
in his laboratory are research associates Rema Balambika,
Mike Churchill, John Offer, and Florian Topert, along with
graduate students John Blankenship and Chris Neidre
The Difficulties of Synthetic Protein Synthesis
Dawson and his colleagues use the technique of solid-phase
synthesis to make the peptides. Invented by Robert Bruce Merrifield
in 1963 (for which he was awarded the 1984 Nobel Prize in
Chemistry), solid phase protein synthesis basically entails
building a peptide step-by-step, starting with a single amino
acid that is attached to a polymer resin. Amino acids are
then added one at a time, the resin is washed between each
successive round, and finally the finished peptide is removed
from the resin.
This basic technique has been improved dramatically through
the years so that today it is used routinely. Some laboratories
have their own automatic peptide synthesis machines, many
research institutions have a core facility that offers peptide
synthesis, and any laboratory in the country can order several
milligrams of purified custom peptides through the mail from
any one of several commercial companies.
Solid phase synthesis does have its limitations, however.
It works well for peptides of around 20 to 25 amino acids
and reasonably well for peptides up to about 50 amino acids.
In some cases, scientists have even been able to synthesize
proteins of around 100 amino acids.
"In general, though," says Dawson, "if you go much above
40 [amino acids], you are going to run into problems."
Not one to see a barrier as a barrier, Dawson has spent
considerable effort to improve the sizes and the yields of
proteins he can routinely make. He uses a number of tricks
that enable him to make proteins and peptides up to about
150 amino acids long. In general, these tricks involve breaking
the sequence up into pieces, synthesizing the pieces one at
a time, and then chemically "ligating" or joining them.
This ligation offers its own unique problems, though, because
of the potential for the reaction to incorrectly join two
peptides. These ligations can join together a carboxy group
with an amino group, but any single unprotected peptide may
have some 10 to 20 of each group, most of them on the side
chains, but the only two that need to be joined are the ones
on the ends. The danger is obtaining a heterogeneous mixture
of branched peptides instead of one uniform pool of linearly
combined peptides. An added challenge is that the reactions
must be done in water at neutral pH.
The solution, says Dawson, has been to modify the peptides
slightly so that the reaction that joins the two ends is highly
favorable and occurs much faster than any other potential
reaction. This is done by making the N terminus of one peptide
a cysteine residue and making the C-terminus of the other
a thioester. The thioester reacts rapidly with the cysteine
side chain and subsequently rearranges to the N-terminus to
form a peptide bond at the site of ligation.
In fact, Dawson says, even in cases where a peptide might
have as many as 14 internal cysteines, it is still possible
to selectively perform reactions and selectively ligate one
peptide to the cysteine at the end of another. However, having
to use cysteines to stitch the peptides together may alter
the sequences of the proteins in undesirable ways, and Dawson
is developing ways of synthesizing proteins with natural sequences
as well.
Now, Dawson and research associate John Offer are using
a modified glycine residue that has the equivalent of a cysteine
side chain to join the two peptides together. "Following that,
we can knock [the cysteine equivalent] off," he says.
This, says Dawson, allows his laboratory to synthesize almost
any protein up to about 150 amino acids. And Dawson and his
laboratory can also combine solid-phase synthesis with biological
expression and folding systems to achieve significantly longer
length proteins.
Post-Translational Modification and HIV Resistance
One applied area in which Dawson and his laboratory work
is the post-translational modification of proteins. Post-translational
modification is a generic term that encompasses many of the
myriad things that happen to a protein in a cell once it is
translated from an mRNA transcript. For example, "phosphorylation"
of proteins is a key element of signaling pathways. However
it is often difficult to generate large quantities of a protein
with a site-specific post-translational modification by biological
methods.
One of the most common post-translational modifications
is the attachment of specific sugars to proteins. In the future,
the Dawson lab hopes to be able to adapt their chemical ligation
techniques to incorporate complex carbohydrates to produce
homogeneous N-linked glycoproteins. Another aim is to understand
the regulation of "palmitoylation" at cysteine residues—a
dynamic lipid modification important for protein localization
in the membrane. Ultimately, he would like to be able to make
any particular site-specific post-translational modifications
he desires.
This expertise in chemically synthesizing proteins also
gives Dawson and his laboratory the ability to routinely make
proteins for a number of applied problems. One problem they
work on is the protease encoded by the human immunodeficiency
virus (HIV).
Dawson recently became involved in a large grant directed
by TSRI Professor Arthur Olson that seeks to establish a drug
design cycle aimed at developing, testing, and refining novel
approaches to making specific inhibitors of HIV protease that
are capable of limiting or eliminating drug resistance. Dawson
is a co-principal investigator on the "Protein Expression
and Analysis" core of the grant, and he provides mutant proteases
and substrate with his synthetic technologies. He also works
with his co-principal investigator, TSRI Professor John Elder,
to look at the substrate specificity of the HIV protease,
working with substrate and substrate-like inhibitors.
Dawson also works with Elder as co-Director of the Protein
Chemistry Core on the Scripps NeuroAIDS Preclinical Studies
center grant, where he applies his synthetic protein chemistry
expertise to the assembly of small proteins, including a variety
of chemokines and cytokines, which are relevant to the study
of neuroAIDS.
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