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)?


"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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TSRI Assistant Professor Philip Dawson, who is a graduate of TSRI's Kellogg School of Science and Technology, wants to produce common and novel protein forms. Photo by Jason S. Bardi.