New Synthetic Protein Could Be Used to Make Artificial Blood

Most proteins are "large" and full of interdependent branches, bends, and pockets in their final folded structure. This complexity aggravates biochemists and protein engineers trying to understand protein structure and function in order to reproduce or create new uses for these natural molecules to combat diseases or for use in industry.

The new type of protein can transport oxygen, similar to human neuroglobin, a molecule that carries oxygen in the brain and peripheral nervous system. In the future, this approach could be used to make artificial blood for use on the battlefield or by emergency-care professionals. The study's findings were published in the March 19, 2009, issue of the journal Nature.

"This is quite a different way of making novel proteins than the rest of the world," stated senior author P. Leslie Dutton, Ph.D., professor of biochemistry and biophysics at the University of Pennsylvania School of Medicine (Penn; Philadelphia, USA). "We've created an unusually simple and relatively small protein that has a function, which is to carry oxygen. No one else has ever done this before."

"Our aim is to design new proteins from principles we discover studying natural proteins," explained coauthor Christopher C. Moser, Ph.D., associate director of the Johnson Foundation at Penn. "For example, we found that natural proteins are complex and fragile and when we make new proteins we want them to be simple and robust. That's why we're not reengineering a natural protein, but making one from scratch."

Currently, protein engineers take an existing biochemical scaffold from nature and change it a bit structurally to make it do something else. "This research demonstrates how we used a set of simple design principles, which challenge the kind of approaches that have been used to date in reproducing natural protein functions," said Dr. Dutton.

The natural design of proteins ultimately lies in their underlying sequence of amino acids; organic compounds that link together to make proteins. In living organisms, this sequence is dictated by the genetic information carried in DNA within chromosomes. This information is then encoded in messenger RNA, which is transcribed from DNA in the nucleus of the cell. The sequence of amino acids for a specific protein is determined by the sequence of nucleotides in messenger RNA. It is the order of the amino acids and the chemical bonds between them that determine how a protein folds into its final shape.

To build their protein, the Penn researchers began with just three amino acids, which code for a helix-shaped column. From this, they assembled a four-column bundle with a loop that resembles a simple candelabrum. They added a heme, a chemical group that contains an iron atom, to bind oxygen molecules. They also added another amino acid called glutamate to add strain to the candelabra to help the columns open up to capture the oxygen. Since heme and oxygen degrade in water, the researchers also designed the exteriors of the columns to deter water to protect the oxygen payload inside.

At first, the team used a synthesizer--a robot that chemically sticks amino acids together in a desired sequence--to make the helix-shaped columns. "We do the first reactions with the robot to figure out the sequence of amino acids that we want," stated Dr. Moser. When they were satisfied with the sequence, they used the bacterium Escherichia coli as a biologic host to make the full protein.

The scientists used chemical tests to validate that their protein did indeed capture oxygen. When the oxygen did bind to the iron heme molecule in the artificial protein, the solution in which the reaction occurred changed color from dark red to scarlet, a color signature almost identical to natural neuroglobin. "This exercise is like making a bus," said Dr. Dutton. "First you need an engine and we've produced an engine. Now we can add other things on to it. Using the bound oxygen to do chemistry will be like adding the wheels. Our approach to building a simple protein from scratch allows us to add on, without getting more and more complicated."

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