Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Engineered Proteins Will Lead to "Synthetic Biology"

08.05.2003


Duke University Medical Center biochemists have developed a computational method to design proteins that can specifically detect a wide array of chemicals from TNT to brain chemicals involved in neurological disorders. In a paper in the May 8, 2003, issue of the journal Nature, they demonstrate the breadth of their design method, and also that such sensor proteins can be re-incorporated into cells to activate cellular signaling and genetic pathways.

The researchers said their achievement constitutes an important step toward a new technology of "synthetic biology," in which scientists construct tailor-made organisms for a variety of tasks, including as "biological sentinels." Such sentinels could find wide use in medical and environmental applications, said the paper’s senior author, Associate Professor of Biochemistry Homme Hellinga, Ph.D.

"You could imagine a bacterium engineered to respond to TNT by fluorescing that could be used to detect mines for humanitarian demining operations," he said. "Also, one might develop a plant whose cells would change its color in response to a pollutant. Such bushes could be planted around chemical facilities to detect groundwater pollution from the facility.



"Such scenarios might sound like something out of science fiction, but they are entirely feasible over the long term using computational design approaches to create sensor proteins," he said.

Hellinga is already developing a glucose-sensing protein that could be used as the basis for a monitor -- and ultimately an artificial pancreas -- for people with diabetes. Also, the TNT-sensing protein is intended to provide free-roaming underwater robots with the capability of "sniffing" a plume of TNT emanating from unexploded ordnance, tracking it to its source, to aid the U.S. Navy’s cleanup program.

The scientists’ research is supported by the Office of Naval Research, the Defense Advanced Research Projects Agency’s "Simulation of Bio-Molecular Microsystems" (SIMBIOSYS) program and the National Institutes of Health.

In the Nature paper, Hellinga and co-authors Loren Looger, Mary Dwyer and James Smith describe how they redesigned proteins from the gut bacterium E. coli. They used "periplasmic binding proteins" which are normally part of the bacterium’s chemical-sensing system by which it detects nutrients. Such protein receptors detect their target molecule via an "active site" with a precise complementary shape and binding properties that fits only that molecule, called a "ligand" -- like a key fitting a lock. Proteins consist of long strings of amino acid units that fold upon themselves to form intricate globular structures.

Basically, the computational design process developed in Hellinga’s laboratory involves redesigning a normal protein’s "lock" to fit a very different molecular key. The computational process narrows down to a manageable few the vast number of possible mutations and their corresponding structures, to fit a particular molecule.

"We call these supra-astronomical calculations, because they can produce more possible combinations than there are particles in the known universe," said Hellinga. "The proteins we work with have binding sites of about 15 amino acid residues. And each of these 15 residues could consist of any of the 20 known amino acids. And we do our calculations in three-dimensional space, so each amino acid is described by a number of different structures that we call rotamers. And we use about 6,000 rotamers to represent the 20 amino acids. So, that alone gives us 6,000 to the 15th power possible sequences."

What’s more, when the researchers factor in the further geometric complexities of how the ligand molecule docks to the active site, said Hellinga, the number of possible designs skyrockets to 10 to the hundredth power possibilities.

However, the Duke biochemists have enhanced and applied a mathematical technique called dead end elimination -- pioneered by mathematician Johan DeSmet of the University of Leuven in Belgium and his colleagues -- to drastically narrow the possible structures. Using this approach, their system can narrow the candidates down to a reasonable number with several days’ worth of calculations on a 30-processor computer network. The high-speed network, called a Beowulf cluster, consists of linked processors of the type available on high-end commercial desktop computers.

In the Nature paper, the scientists described adapting E. coli proteins to detect three very different molecules of environmental and clinical importance.

-- TNT, which is a nonbiological compound and carcinogen that the Navy is seeking to detect, as part of developing a TNT-sensing robot to aid its environmental cleanup effort.

-- lactate, which is an indicator of metabolic stress in the body and also associated with certain cancers. Thus, a lactate-sensing protein could locate metabolic distress in the body. Also, lactate has a "left-handed" and "right-handed" molecular form. Demonstrating that an engineered protein could distinguish such "chiral" forms would be of great interest to pharmaceutical firms, said Hellinga, because they must purify drugs to eliminate unwanted chiral forms that can be highly toxic.

-- serotonin, which is a neurotransmitter molecule that nerve cells use to trigger nerve impulses in neighboring cells. Fluctuating serotonin levels are associated with certain psychiatric disorders and elevated levels are indicative of certain bowel tumors.

Also, building a serotonin-sensing protein is a special design challenge, said Hellinga, because the molecule has distinct regions that are negatively or positively charged. "And frankly, we thought it was a rather dramatic achievement to build a receptor for a neurotransmitter from a bacterial protein," said Hellinga.

"We basically tried to show that this is a very general design approach," he said. "We demonstrate that, within reason, no matter what the chemical nature of the ligand, we can design an active site to specifically bind it." Also to demonstrate the adaptability of their technique, the researchers started with a number of different E. coli proteins in their design effort.

They applied their computational method to automatically generate a collection of possible designs and to narrow them down through dead end elimination to arrive at a workable number of candidates.

Then, the scientists actually constructed a selection of the candidate proteins by genetically altering E. coli to produce them. To measure the proteins’ sensing capabilities, they used a technology that they had developed earlier for the periplasmic binding proteins, and engineered the molecules to incorporate a fluorescent molecule, called a fluorophore, attached such that the protein would only fluoresce if the target molecule had plugged into the protein.

The resulting sensor proteins proved in test tube studies to have high affinity and specificity for their target molecules, said Hellinga. For example, he said, both the TNT-sensing and serotonin-sensing proteins could not be fooled by "decoy" molecules that were very closely related to TNT or serotonin in structure. Also, the lactate-detecting protein preferentially bound only to one chiral form of the molecule, proving that the design system could produce such specific sensing proteins.

The researchers used a feedback technique, called "quantitative structure-activity relationship," (QSAR) to apply data on molecular interactions in the experimental proteins to optimize the theoretical design calculations even further.

Finally, Hellinga and his colleagues sought to demonstrate that the sensor proteins could function in the machinery of living cells.

"We had shown that these proteins could function as sensors in the test tube by labeling them with fluorophores," said Hellinga. "But we were also interested in putting them back into a biological system and showing that we could modulate the behavior of that biological system. It’s an important step toward developing what we call ’synthetic biology.’"

Thus, the scientists showed that they could insert either the TNT-binding protein or the lactose-binding protein into E. coli bacteria in such a way as to activate a "reporter gene," which signaled that their molecule had become a functioning component of the bacterial genetic machinery. Such experiments proved the importance of their new design approach, said Hellinga.

"The potential implications of this technique are very broad," he said. "Essentially what we have done is establish control over molecular recognition of small molecules in biology. Such recognition is central to the most basic processes in organisms, and we have demonstrated that not only can we design highly specific receptors, but we can put them into biological systems to control them.

"Also, we have shown that we can create sensors that are exquisitely sensitive, even to the chiral forms of molecules. Such sensors could prove invaluable to pharmaceutical companies, since many drugs are chiral -- with one form being pharmacologically active and the other being either inactive or toxic."

According to Hellinga, the Duke computational design system is far superior to the other major technique -- called "directed evolution" -- used to tailor proteins to bind specific molecules. In directed evolution, researchers create a large library of candidate proteins and screen them for affinity and specificity to a target molecule.

"On the one hand directed evolution is a very elegant method, in which they’ve essentially programmed nature to give the right answer," said Hellinga. "But there are real limitations on how large a variety of possible protein structures can be surveyed. Our computational methods can overcome that limitation."

In terms of commercial applications, he said, the university is exploring the launching of a company to market and support the design methodology and applications developed by Hellinga and his colleagues.

Dennis Meredith | Duke University
Further information:
http://dukemednews.org/news/article.php?id=6549

More articles from Life Sciences:

nachricht Symbiotic bacteria: from hitchhiker to beetle bodyguard
28.04.2017 | Johannes Gutenberg-Universität Mainz

nachricht Nose2Brain – Better Therapy for Multiple Sclerosis
28.04.2017 | Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik IGB

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Making lightweight construction suitable for series production

More and more automobile companies are focusing on body parts made of carbon fiber reinforced plastics (CFRP). However, manufacturing and repair costs must be further reduced in order to make CFRP more economical in use. Together with the Volkswagen AG and five other partners in the project HolQueSt 3D, the Laser Zentrum Hannover e.V. (LZH) has developed laser processes for the automatic trimming, drilling and repair of three-dimensional components.

Automated manufacturing processes are the basis for ultimately establishing the series production of CFRP components. In the project HolQueSt 3D, the LZH has...

Im Focus: Wonder material? Novel nanotube structure strengthens thin films for flexible electronics

Reflecting the structure of composites found in nature and the ancient world, researchers at the University of Illinois at Urbana-Champaign have synthesized thin carbon nanotube (CNT) textiles that exhibit both high electrical conductivity and a level of toughness that is about fifty times higher than copper films, currently used in electronics.

"The structural robustness of thin metal films has significant importance for the reliable operation of smart skin and flexible electronics including...

Im Focus: Deep inside Galaxy M87

The nearby, giant radio galaxy M87 hosts a supermassive black hole (BH) and is well-known for its bright jet dominating the spectrum over ten orders of magnitude in frequency. Due to its proximity, jet prominence, and the large black hole mass, M87 is the best laboratory for investigating the formation, acceleration, and collimation of relativistic jets. A research team led by Silke Britzen from the Max Planck Institute for Radio Astronomy in Bonn, Germany, has found strong indication for turbulent processes connecting the accretion disk and the jet of that galaxy providing insights into the longstanding problem of the origin of astrophysical jets.

Supermassive black holes form some of the most enigmatic phenomena in astrophysics. Their enormous energy output is supposed to be generated by the...

Im Focus: A Quantum Low Pass for Photons

Physicists in Garching observe novel quantum effect that limits the number of emitted photons.

The probability to find a certain number of photons inside a laser pulse usually corresponds to a classical distribution of independent events, the so-called...

Im Focus: Microprocessors based on a layer of just three atoms

Microprocessors based on atomically thin materials hold the promise of the evolution of traditional processors as well as new applications in the field of flexible electronics. Now, a TU Wien research team led by Thomas Müller has made a breakthrough in this field as part of an ongoing research project.

Two-dimensional materials, or 2D materials for short, are extremely versatile, although – or often more precisely because – they are made up of just one or a...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Fighting drug resistant tuberculosis – InfectoGnostics meets MYCO-NET² partners in Peru

28.04.2017 | Event News

Expert meeting “Health Business Connect” will connect international medical technology companies

20.04.2017 | Event News

Wenn der Computer das Gehirn austrickst

18.04.2017 | Event News

 
Latest News

Wireless power can drive tiny electronic devices in the GI tract

28.04.2017 | Medical Engineering

Ice cave in Transylvania yields window into region's past

28.04.2017 | Earth Sciences

Nose2Brain – Better Therapy for Multiple Sclerosis

28.04.2017 | Life Sciences

VideoLinks
B2B-VideoLinks
More VideoLinks >>>