Caltech Researchers Show How Organic Carbon Compounds Emitted by Trees Affect Air Quality

Research provides first-ever glimpse of role of epoxides in atmospheric chemistry

PASADENA, Calif.—A previously unrecognized player in the process by which gases produced by trees and other plants become aerosols—microscopically small particles in the atmosphere—has been discovered by a research team led by scientists at the California Institute of Technology (Caltech).

Their research on the creation and effects of these chemicals, called epoxides, is being featured in this week's issue of the journal Science.

Paul Wennberg, the R. Stanton Avery Professor of Atmospheric Chemistry and Environmental Science and Engineering and director of the Ronald and Maxine Linde Center for Global Environmental Science at Caltech, and John Seinfeld, the Louis E. Nohl Professor and professor of chemical engineering, have been studying the role of biogenic emissions—organic carbon compounds given off by plants and trees—in the atmospheric chemical reactions that result in the creation of aerosols.

"If you mix emissions from the city with emissions from plants, they interact to alter the chemistry of the atmosphere," Wennberg notes.

While there's been plenty of attention paid to the effect of emissions from cars and manufacturing, less is understood about what happens to biogenic emissions, especially in places where there are relatively few man-made emissions. That situation is the focus of the research that led to this Science paper. "What we're interested in," Wennberg explains, "is what happens to the chemicals produced by trees once they are emitted into the atmosphere."

In these studies, the research team focused on a chemical called isoprene, which is given off by many deciduous trees. "The king emitters are oaks," Wennberg says. "And the isoprene they emit is one of the reasons that the Smoky Mountains appear smoky."

Isoprene is no minor player in atmospheric chemistry, Wennberg notes. "There is much more isoprene emitted to the atmosphere than all of the gases—gasoline, industrial chemicals—emitted by human activities, with the important exceptions of methane and carbon dioxide," he says. "And isoprene only comes from plants. They make hundreds of millions of tons of this chemical . . . for reasons that we still do not fully understand."

"Much of the emission of isoprene occurs where anthropogenic emissions are limited," adds Caltech graduate student Fabien Paulot, the paper's first author. "The chemistry is very poorly understood."

Once released into the atmosphere, isoprene gets "oxidized or chewed on" by free-radical oxidants such as OH, explains Wennberg. It is this chemistry that is the focus of this new study. In particular, the research was initiated to understand how the oxidation of isoprene can lead to formation of atmospheric particulate matter, so-called secondary organic aerosol. "A small fraction of the isoprene becomes secondary organic aerosol," Seinfeld notes, "but because isoprene emissions are so large, even this small fraction is important."

Up until now, the chemical pathways from isoprene to aerosol were not known. Wennberg, Seinfeld, and their colleagues discovered that this aerosol likely forms from chemicals known as epoxides.

The name is apt. "These epoxides are nature's glue," says Wennberg. And, much like the epoxy you buy in a hardware store—which requires the addition of an acid for the compound to turn into glue—the epoxides found in the atmosphere also need an acidic kick in order to become sticky.

"When these epoxides bump into particles that are acidic, they make glue," Wennberg explains. "The epoxides precipitate out of the atmosphere and stick to the particles, growing them and resulting in lowered visibility in the atmosphere." Because the acidity of the aerosols is generally higher in the presence of anthropogenic activities, the efficiency of converting the epoxides to aerosol is likely higher in polluted environments, illustrating yet another complex interaction between emissions from the biosphere and from humans. 

"Particles in the atmosphere have been shown to impact human health, as they are small enough to penetrate deep into the lungs of people. Also, aerosols impact Earth's climate through the scattering and absorption of solar radiation and through serving as the nuclei on which clouds form. So it is important to know where particles come from," notes Seinfeld.

The research team was able to make this scientific leap forward thanks to their development of a new type of chemical ionization mass spectrometry (CIMS), led by coauthor and Caltech graduate student John Crounse. "These new CIMS methods open up a very wide range of possibilities for the study of new sets of compounds that scientists have been largely unable to measure previously, mainly because they decompose when analyzed with traditional techniques."

In general, molecules identified and quantified using mass spectroscopy must first be converted to charged ions. They are then directed into an electric field, where the ions are sorted by mass. The problem with traditional ionization techniques is that delicate molecules, such as those produced in the oxidation of isoprene, generally fragment during the ionization process, making their identification difficult or impossible. "This new method was originally developed in order to allow scientists to make atmospheric measurements from airplanes. It is able to ionize gases, even fragile peroxide compounds, while still preserving information about the size or mass of the original molecule," says Wennberg.

That makes determining the individual gases in a complex mixture much easier—especially when, as it turned out, you're looking at a chemical you weren't expecting to find.

Wennberg and colleagues also used oxygen isotopes—oxygen atoms with different numbers of neutrons in their nucleus, and thus different masses—to gain insight into the chemical mechanism yielding epoxides. Epoxides have remained unindentified so far because they have the same mass as another chemical that had been anticipated to form in isoprene oxidation, peroxide. "The oxygen isotopes separated the peroxides from epoxides and further showed that as the epoxides form, OH is recycled to the atmosphere," comments Paulot. "Since OH is the atmosphere detergent, cleaning the atmosphere of many chemicals, the recycling has important implications for the overall oxidizing capacity of the atmosphere."

The identification of a major photochemical pathway to formation of epoxides helps to explain just how tree emissions of organic carbon compounds influence the air in both city and rural settings. While trees aren't exactly the "killers" that Ronald Reagan was once so famously derided for calling them, their isoprene emission levels can—and often probably should—"be a part of the criteria we use when buying and planting trees in a polluted urban setting," notes Wennberg. In fact, he points out, the South Coast Air Quality Management District in Southern California already does this with its list of "approved" trees that don't emit large amounts of organic carbon compounds into the atmosphere.

In addition to Wennberg, Paulot, Crounse, and Seinfeld, other authors on the Science paper, "Unexpected epoxide formation in the gas-phase photooxidation of isoprene," are Henrik Kjaergaard of the University of Otago in New Zealand and the University of Copenhagen in Denmark; former Caltech postdoctoral scholar Andreas Kürten, now at Goethe University in Germany; and Caltech postdoctoral scholar Jason St. Clair.

Purchase of the mass spectrometer used in this study was funded by a Major Research Instrumentation Award from the National Science Foundation. Additional support for the work described in the Science article came from Caltech trustee William Davidow and by grants from the Office of Science, the U.S. Department of Energy, the U.S. Environmental Protection Agency, the Royal Society of New Zealand, and NASA.

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Caltech Chemists Say Antibody Surrogates Are Just a "Click" Away

Chemists at the California Institute of Technology (Caltech) and the Scripps Research Institute have developed an innovative technique to create cheap but highly stable chemicals that have the potential to take the place of the antibodies used in many standard medical diagnostic tests.

James R. Heath, the Elizabeth W. Gilloon Professor and professor of chemistry, along with K. Barry Sharpless, the W. M. Keck Professor of Chemistry at the Scripps Research Institute and winner of the 2001 Nobel Prize in Chemistry, and their colleagues, describe the new technique in the latest issue of Angewandte Chemie, the leading European journal of chemistry.

Last year, Heath and his colleagues announced the development of the Integrated Blood-Barcode Chip, a diagnostic medical device, about the size of a microscope slide, which can separate and analyze dozens of proteins using just a pinprick of blood. The barcode chip employed antibodies, proteins utilized by the immune system to identify, bind to, and remove particular foreign compounds, such as bacteria and viruses-or other proteins.

"The thing that limits us in being able to go to, say, 200 proteins in the barcode chip is that the antibodies that you use to detect the proteins are unstable and expensive," says Heath. "We have been frustrated with antibodies for a long time, so what we wanted to be able to do was develop antibody equivalents-what we call 'protein capture agents'-that can bind to a particular protein with very high affinity and selectivity, and that pass the following test: you put a powder of them in your car trunk in August in Pasadena, and you come back a year later and they still work."

In the new work, Heath and his colleagues, including Caltech graduate student Heather D. Agnew, the first author on the Angewandte paper, have developed a protocol to quickly and cheaply make such highly stable compounds, which are composed of short chains of amino acids, or peptides. "I actually traveled to Chicago with a vial of my capture agents as airline carry-on luggage, and came back with it, and the reagent still worked," says Agnew.

The technique makes use of the "in situ click chemistry" method, introduced by Sharpless in 2001, in which chemicals are created by joining-or "clicking"-smaller subunits together.

To create a capture agent for a particular protein, the scientists devised a stepwise approach in which the first subunit of the capture agent is identified, and that unit, plus the protein, is used to identify the second subunit, and so on. For the first subunit, a fluorescent label is added to the protein, which is then incubated with a bead-based library of tens of millions of short-chain peptides, representing all the potential building blocks for the capture agent. When one of those peptides binds to the protein of interest, the fluorescent label is visualized on the bead (red, blue, or green, depending on the type of label), allowing the linked protein-peptide complex to be identified.

That first peptide-which is about a third of the length of the final capture agent the scientists are trying to make-is then isolated, purified, and modified on one end by the addition of a chemical group called an alkyne. This is the anchor peptide, which is then incubated, together with the same protein, with the bead-based library. The bead-based library now contains peptides that have been chemically modified to contain an azide group at one end. The alkyne group on the added peptide can potentially chemically react with the azide group of the library's peptides, to create a new peptide that is now two segments long.

However, the reaction can only occur when the second peptide comes into close contact with the first on the surface of the target protein, which means that both must have  affinity for that protein; essentially, the protein itself builds an appropriate capture agent. The two-segment-long peptide is then isolated and purified, "and then we modify the end of THAT with an alkyne, and add it back to the library, to produce a three-segment peptide, which is long enough to be both selective for and specific to the target protein," Heath says.

"What Heath has shown now is that in several iterations, a high-affinity ligand for a protein can be created from blocks that do not bind to the protein all that well; the trick is to repeat the in situ screen several times, and the binding improves with every iteration," Sharpless says.

"This is about as simple a type of chemistry as you can imagine," says Heath. The process, he says, makes "trivial" the "Herculean task of finding molecules that bind selectively and with high affinity to particular proteins. I see no technical reason it couldn't replace any antibody."

The paper, "Iterative in situ Click Chemistry Creates Antibody-Like Protein Capture Agents," was published in the June 22 issue of Angewandte Chemie, and highlighted in an editorial in the June issue of Nature Chemistry. The other coauthors are, at Caltech, Rosemary D. Rohde, Steven W. Millward, Arundhati Nag, Woon-Seok Yeo, Abdul Ahad Tariq, Russell J. Krom, and Vanessa M. Burns; and, at the Scripps Research Institute, Jason E. Hein, Suresh M. Pitram, and Valery V. Fokin.

The work at Caltech was funded by the National Cancer Institute and by a subcontract from the MITRE Corporation.

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Caltech Researchers Explore How Cells Reconcile Mixed Messages in Decisions About Growth

Findings have implications for tissue engineering, understanding of tumor development

PASADENA, Calif.—The cells in our body are constantly receiving mixed messages. For instance, an epithelial cell might be exposed to one signal telling it to divide and, simultaneously, another telling it to stop dividing. Understanding the process by which these competing environmental cues are reconciled—as well as understanding the cues themselves—might allow bioengineers to promote tissue growth when and where it's needed, and to discourage it when and where it's not.

The tug-of-war between these two sets of influences, and the effects they have on tissue growth, are explained and explored in a paper authored by scientists from the California Institute of Technology (Caltech) and published online in the early edition of the Proceedings of the National Academy of Sciences (PNAS). The findings in the paper may have implications for our understanding of how cancer develops, as well as for how best to grow tissues in a laboratory.

In normal epithelial tissues, mature cells that are in contact with one another tend not to divide, explains Anand Asthagiri, assistant professor of chemical engineering at Caltech, and the paper's principal investigator. This process, known as contact inhibition, is one of the ways the body keeps cell growth in check. When contact inhibition is disrupted, you get uncontrolled growth and the formation of tumors.

But what Asthagiri and colleagues have found is that contact inhibition is not a "master switch" that overrides all other environmental signals. The human body is, after all, a complex environment. And in that complex environment, contact inhibition doesn't—can't—work by itself. It is instead part of what Asthagiri calls a "tunable system," one that takes into account, and is influenced by, other signals. Among those are growth signals such as epidermal growth factor (EGF).

When Asthagiri and his colleagues studied the interplay between contact inhibition and EGF in groups of epithelial cells, they found that the cells have a threshold of sensitivity to EGF. If EGF levels dip below the threshold, contact inhibition takes hold and puts the brakes on cell division. But if EGF levels rise above the threshold, it overrides the effects of cell-cell contact and promotes cell division and tissue growth.

Both factors can potentially be manipulated—either to raise or lower the levels of growth factor or, as Asthagiri and colleagues showed in their paper, to raise or lower the contact-inhibition threshold.

In other words, Asthagiri explains, the team's research showed that it's possible to tune the system—to make cells more or less able to respond to a certain level of EGF by "playing with the extent of the contact the cells have with their neighbors."

One way to do that is to crowd the cells. "For instance," he says, "if you take a large number of cells and force them into the same area in which only a few cells are normally found, the cells become somewhat deaf to the growth factors. In order to get these cells to divide, you really have to crank up the level of growth factors they're exposed to."

You can achieve a similar result, Asthagiri adds, by creating cells that overexpress a protein called E-cadherin, which is a tumor suppressor protein that promotes adhesion of one cell to another. "This makes the cells less willing to divide," he notes, "which means they need a higher level of growth factor before they will divide."

The relationships between these competing influences "are really striking when you let them play out" under the influence of cell geography, says Asthagiri—that is, when the cells grow as a multicellular cluster. The reality is that not all cells in a cluster are exposed to the same amount of inhibition. For instance, the cells in the center of the group—pressed against other cells on all sides—will experience more contact, and will require a larger amount of growth factor if they are to overcome that inhibiting signal. The cells on the periphery of a cluster, on the other hand, get a relative whisper of an inhibitory signal; it doesn't take nearly as much growth factor to prompt those cells to divide.

Thus, it's possible to find a level of growth factor that will override the contact inhibition signal only for the peripheral cells, and then to find a second level that will allow division throughout the cluster. In other words, says Asthagiri, "You can tune the system; you can make the periphery grow more quickly relative to the rest of the area, or you can get the entire cluster to increase in size all at once."

"This is useful," he adds, "in thinking about how to engineer organs and tissues. I believe that this can become an important building block, a part of the tool set, that allows us to grow multicellular structures—and, ultimately, tissues—in specific, spatial ways."

And as for cancer? It's long been assumed that contact inhibition acts as a sort of switch that, when present, prevents tumor formation and, when absent, results in cell overgrowth and cancer. "Our findings support a more graded perspective of contact inhibition," the researchers write in the PNAS paper. Keeping in mind that cancer is often the result of an accumulation of genetic damage, they say, it seems likely that each "hit" to a cell's DNA might subtly lower the threshold at which EGF is capable of overriding contact inhibition to promote unbridled cell division and tumor growth.

"This tunability of the threshold amount of EGF," the researchers write, "would seem to be a fragility in cell cycle regulation that is exploited during cancer development."

Asthagiri's coauthors on the PNAS paper, "Tunable interplay between epidermal growth factor and cell-cell contact governs the spatial dynamics of epithelial growth," include Caltech graduate students Jin-Hong Kim, the paper's first author, and Keiichiro Kushiro, as well as former Caltech graduate student Nicholas A. Graham, who is now a postdoctoral fellow at the Crump Institute for Molecular Imaging at UCLA.

The work described was supported by the Concern Foundation for Cancer Research and the Jacobs Institute for Molecular Engineering for Medicine at Caltech.

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Harry Gray Awarded for Lifetime of Basic Research in Chemistry and Advances in Solar Fuel

For decades of breakthroughs in bioinorganic and inorganic photochemistry powering his current work in renewable fuels, Harry Gray, the Beckman Professor of Chemistry and founding director of the Beckman Institute at Caltech, has been named the recipient of the 2009 Welch Award in Chemistry. The award is presented annually by the Houston-based Welch Foundation for lifetime achievement in basic research.

"Harry Gray is a gifted researcher, teacher, and statesman for chemistry," said Dennis Hendrix, chairman of the Welch Foundation. "He has touched almost every aspect of inorganic chemistry in his 45-year career and helped cofound the fields of biological inorganic chemistry and inorganic photochemistry."

Early in his career, Gray developed ligand field theory of inorganic electronic structures, insights still widely used today. He found that the bonding models he had developed for inorganic substances also were useful in understanding many biological processes, leading to the creation of the new field of biological inorganic chemistry.

He moved on to study electron transfer, respiration, and photosynthesis. In the early 1980s, his group made a major discovery when they found that molecules do not have to be in close contact to transfer electrons as previously thought, but instead two metal atoms could complete the transfer over "long" distances of as much as two or three nanometers and across 20 atoms or more. This discovery is significant in that the longer distances provide the opportunity to capture and store the energy created by the electron moving from one molecule to the next, rather than simply generating heat that is wasted in the "short" distance transfers.

This breakthrough forms the basis for photosynthetic systems that can store sunlight's energy as a chemical fuel. The fuel then can be used to make electricity when needed. His current work is exploring how best to duplicate nature's photosynthesis, the process by which plants turn sunlight into food and concurrently produce the oxygen essential to life. Gray and his team are exploring the use of abundant inorganic (nonliving) materials and sunlight to generate hydrogen fuel and clean water economically on a large scale.

The Welch Foundation supports science through research and departmental grants, funding of academic chairs, an annual chemical conference and support for other chemistry-related programs. Gray will receive the award in October at a banquet hosted by the Welch Foundation in Houston. At that time, he will be presented with the Welch Award gold medallion and the $300,000 prize.

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New Radiation-free Targeted Therapy Detects and Eliminates Breast-Cancer Tumors in Mice

Caltech researcher helps develop technique that homes in on aggressive, difficult-to-treat HER2+ breast cancer cells

PASADENA, Calif.--Combining a compound known as a gallium corrole with a protein carrier results in a targeted cancer therapy that is able to detect and eliminate tumors in mice with seemingly fewer side effects than other breast-cancer treatments, says a team of researchers from the California Institute of Technology (Caltech), the Israel Institute of Technology (Technion) and the Cedars-Sinai Medical Center.

A paper describing their work is highlighted in this week's issue of the online edition of the Proceedings of the National Academy of Sciences (PNAS).

Corroles have very similar structures to the porphyrin molecules used in a well-studied cancer treatment known as photodynamic therapy, or PDT, in which porphyrin compounds injected into the body are exposed to specific wavelengths of laser light. The light prompts the porphyrins to produce active, tumor-killing oxygen radicals.

The difference between porphyrins and corroles, says Harry Gray, Caltech's Arnold O. Beckman Professor of Chemistry and founding director of the Beckman Institute, is that some corroles don't require a laser boost to turn lethal. "The striking thing about gallium corroles is that they apparently kill cancer cells in the dark," says Gray. "We don't yet know exactly how this works, but what we've seen so far tells us that it does work."

He notes that "ongoing work in our laboratories focuses on testing our leading hypotheses for elucidating the mechanism of action."

In the experiments described in the PNAS paper, the team paired a gallium corrole with a carrier protein, then aimed it at cells that carry the human epidermal growth factor receptor 2 (HER2). The presence of a HER2 receptor is the hallmark of about 25 percent of breast cancers, and marks those tumors as particularly aggressive and difficult to treat.

In trials in mice, the targeted corrole was able to shrink tumors at doses five times lower than that of the standard chemotherapeutic agent for HER2-positive tumors, a drug called doxorubicin. In contrast with doxorubicin, the corrole was injected into the bloodstream, rather than directly into the tumor.

"We looked at three groups of mice with human tumors," explains paper coauthor Lali Medina-Kauwe, an assistant professor of medicine at the David Geffen School of Medicine at UCLA, and a faculty research scientist in the Department of Biomedical Science at Cedars-Sinai Medical Center in Los Angeles. "In one, we introduced just the protein carrier, without the corrole; tumor growth in those mice did not change. In other mice, we gave the corrole without the carrier protein; this led to some tumor suppression. But it was the last group, the ones that got the corrole with the carrier protein, that experienced the most therapeutic effect."

"The fact that we can target this compound means we can give it at very low concentrations," adds coauthor Daniel Farkas, director of Cedar-Sinai's Minimally Invasive Surgical Technologies Institute. "Using lower concentrations means less toxicity. Doxorubicin tends to have significant heart toxicity; this therapy seems likely to be much less damaging to the heart."

In addition, adds Medina-Kauwe, targeted compounds can seek out tumors wherever they may be. "One of the beauties of targeting," she says, " is that we can go after metastatic tumors that are too small to be seen."

These targeted gallium corroles are not only effective, they're also easy to study, notes Zeev Gross, the Reba May & Robert D. Blum Academic Chair at Technion, the Israeli Institute of Technology, in Haifa, and another of the paper's coauthors. "In most cases, if people want to get a closer look at a drug in vivo, they have to attach a fluorescent probe to it--and that turns it into a different molecule. But in our case, the active molecule we're tracking does the fluorescing. We get to track the original, unmodified molecule and are hence able to follow its distribution among different organs in live animals."

The difficulty in getting to this point, notes Gray, is that corroles have been challenging to synthesize. "Then Zeev came up with a powerful synthetic method to make corroles," he says. "We went from being able to make a couple of milligrams in two years to being able to make two grams in two days. It really puts corroles on the map."

Gray and Gross further add, "It is truly fulfilling to see how the close collaboration between our research groups at Caltech and the Technion, which started 10 years ago with a focus on developing the fundamental science of corroles, led to pharmaceutical utility when we joined forces with Medina-Kauwe and Farkas, who are experts in cellular biology and biomedical imaging technologies."

The work described in the paper, "Tumor detection and elimination by a targeted gallium corrole," was supported by grants from the National Science Foundation, the National Institutes of Health, the U.S. Department of Defense, Susan G. Komen for the Cure, the Donna and Jesse Garber Award, the Gurwin Foundation, the United States-Israel Binational Science Foundation, and by the U.S. Navy Bureau of Medicine and Surgery.

In addition to the researchers mentioned above, the other scientists contributing to this work include senior scientist Atif Mahammed from Technion, and research associates Hasmik Agadjanian and Altan Rentsendorj, postdoc Vinod Valluripalli, and graduate students Jun Ma and Jae Youn Hwang from Cedars-Sinai.

# # #

Contact:    Lori Oliwenstein
        (626) 395-3631
        lorio@caltech.edu

Visit the Caltech Media Relations website at http://pr.caltech.edu/media.

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Caltech Scientists Create New Enzymes for Biofuel Production

Enzymes are important step toward cheaper biofuels

Researchers at the California Institute of Technology (Caltech) and world-leading gene-synthesis company DNA2.0 have taken an important step toward the development of a cost-efficient process to extract sugars from cellulose--the world's most abundant organic material and cheapest form of solar-energy storage. Plant sugars are easily converted into a variety of renewable fuels such as ethanol or butanol.

In a paper published this week in the early edition of the Proceedings of the National Academy of Sciences, Frances H. Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering and Biochemistry at Caltech, and her colleagues report the construction of 15 new highly stable fungal enzyme catalysts that efficiently break down cellulose into sugars at high temperatures. Previously, fewer than 10 such fungal cellobiohydrolase II enzymes were known. In addition to their remarkable stabilities, Arnold's enzymes degrade cellulose over a wide range of conditions.

Biofuels are made by converting renewable materials--for example, corn kernels, wood chips left over from pulp and paper production, prairie grasses, and even garbage--into fuels and chemicals. Most biofuels used today are made from the fermentation of starch from corn kernels. That process, although simple, is costly because of the high price of the corn kernels themselves.

Agricultural waste, such as corn stover (the leaves, stalks, and stripped cobs of corn plants, left over after harvest), is cheap. These materials are largely composed of cellulose, the chief component of plant-cell walls. Cellulose is far tougher to break down than starch. An additional complication is that while the fermentation reaction that breaks down corn starch needs just one enzyme, the degradation of cellulose requires a whole suite of enzymes, or cellulases, working in concert.

The cellulases currently used industrially, all of which were isolated from various species of plant-decaying filamentous fungi, are both slow and unstable, and, as a result, the process remains prohibitively expensive. "Even a two-fold reduction in their cost could make a big difference to the economics of renewable fuels and chemicals," says Arnold.

Arnold and Caltech postdoctoral scholar Pete Heinzelman created the 15 new enzymes using a process called structure-guided recombination. Using a computer program to design where the genes recombine, the Caltech researchers "mated" the sequences of three known fungal cellulases to make more than 6,000 progeny sequences that were different from any of the parents, yet encoded proteins with the same structure and cellulose-degradation ability.

By analyzing the enzymes encoded by a small subset of those sequences, the Caltech and DNA2.0 researchers were able to predict which of the more than 6,000 possible new enzymes would be the most stable, especially under higher temperatures (a characteristic called thermostability).

Thermostability is a requirement of efficient cellulases, because at higher temperatures--say, 70 or even 80 degrees Celsius--chemical reactions are more rapid. In addition, cellulose swells at higher temperatures, which makes it easier to break down. Unfortunately, the known cellulases from nature typically won't function at temperatures higher than about 50 degrees Celsius.

"Enzymes that are highly thermostable also tend to last for a long time, even at lower temperatures," Arnold says. "And, longer-lasting enzymes break down more cellulose, leading to lower cost."

Using the computer-generated sequences, coauthor Jeremy Minshull and colleagues from DNA2.0 of Menlo Park, California, synthesized actual DNA sequences, which were transferred into yeast in Arnold's laboratory. The yeast produced the enzymes, which were then tested for their cellulose-degrading ability and efficiency. Each of the 15 new cellulases reported in the PNAS paper was more stable, worked at significantly higher temperatures (70 to 75 degrees Celsius), and degraded more cellulose than the parent enzymes at those temperatures.

"This is a really nice demonstration of the power of synthetic biology," Arnold says. "You can rapidly generate novel, interesting biological materials in the laboratory, and you don't have to rely on what you find in nature. We just emailed DNA2.0 sequences based on what we pulled out of a database and our recombination design, and they synthesized the DNA. We never had to go to any organism to get them. We never touched a fungus."

Next, the researchers plan to use the structure-guided recombination process to perfect each of the half-dozen or so cellulases that make up the soup of enzymes required for the industrial degradation of cellulose. "We've demonstrated the process on one of the components. Now we have to create families of all of the other components, and then look for the ideal mixtures for each individual application," Arnold says, with the ultimate goal of creating a cost-efficient recipe for cellulosic biofuel.

"If you think about it, energy is the biggest industry there is," Arnold says. "If we can replace foreign oil with renewable biofuels, that's an enormous contribution. And that replacement is slow right now because these enzymes are just too expensive."

The work in the paper, "A Family of Thermostable Fungal Cellulases Created by Structure-Guided Recombination," was supported by the Army-Industry Institute for Collaborative Biotechnologies and the Caltech Innovation Institute.

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Caltech and UCSD Researchers Shed Light on How Proteins Find Their Shapes

The researchers bring together theoretical models and experimental data to explain protein folding

PASADENA, Calif.--Researchers from the California Institute of Technology (Caltech) and the University of California at San Diego (UCSD) have brought together UCSD theoretical modeling and Caltech experimental data to show just how amino-acid chains might fold up into unique, three-dimensional functional proteins.

Their insights were recently published in the February 10 issue of the Proceedings of the National Academy of Sciences (PNAS).

The paper details the matching of a series of protein-folding models created by the UCSD team (led by Peter Wolynes, UCSD professor of chemistry and biochemistry and physics) with experimental data gathered using a novel technique created by the Caltech team (led by Faculty Associate in Chemistry Jay Winkler and Harry Gray, Caltech's Arnold O. Beckman Professor of Chemistry and founding director of the Beckman Institute).

The Winkler-Gray method of watching proteins as they crumple and fold involves the use of a picosecond camera that captures fluorescent flashes as a laser pulse excites a donor probe, which emits light and transfers that light to an acceptor probe. The distance between the donor and acceptor change as the amino-acid chain transforms itself into a three-dimensional protein.

In the PNAS paper, the two groups combined the Caltech experimental technique--first described in a 2002 paper published in the Journal of the American Chemical Society--with Wolynes's protein-folding models to see if they could come up with the precise folding pattern of cytochrome c, a protein that is part of the mitochondrial electron-transfer chain that turns food into cellular energy.

At first the models and the experimental data seemed to be describing two entirely different things, according to Winkler. "The researchers had to account for charge-charge interactions between amino acids that appear to be important--the way that like charges repel and opposite charges attract," he explains. "And they had to consider the hydrophobic interactions--the way that oily parts of the proteins like to stick together but are repelled by the watery parts. When their models took account of these interactions, it fit the experimental data."

"It was the first time anyone has been able to develop a theoretical model able to account for the results we've been getting with our time-resolved energy-transfer experiments," adds Gray.

Other coauthors on the PNAS paper, entitled "Electrostatic effects on funneled landscapes and structural diversity in denatured protein ensembles," are Patrick Weinkam from UCSD and Ekaterina Pletneva, formerly at Caltech and now at Dartmouth College.

This work was supported by grants from the National Institutes of Health and by a National Science Foundation Center for Theoretical Biological Physics grant.

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Self-Regulating Molecular "Transformers" Control the Intracellular Delivery of Proteins

PASADENA, Calif.--Scientists from the California Institute of Technology (Caltech) have uncovered the Transformer like properties of molecules responsible for carrying and depositing proteins to their correct locations within cells. The research could eventually lead to novel treatments for diseases that result from flaws in protein delivery as well as the development of new types of antibiotics.

Shu-ou Shan, an assistant professor of chemistry at Caltech, and her colleagues looked specifically at a pair of proteins that sort cellular proteins and deliver them to their destinations--a process that is essential for establishing and maintaining cellular organization. The proteins, known as the signal recognition particle (SRP) and the SRP receptor (SR), are responsible for shuttling more than a third of all cellular proteins to their targets, including the insulin protein. The SRP/SR system is present in all three kingdoms of life, from humans and other animals, to plants and fungi, to bacteria and primitive archaean organisms.

By tracking the movement of fluorescently tagged molecules, Shan and her colleagues were able to track the behavior of SRP and SR during the protein pick-up and delivery process.

They found that the binding of a protein cargo by the SRP molecule triggered the accelerated assembly of a molecular complex containing SRP, the cargo, and the SR protein. The SRP-SR complex then delivered the cargo to the cell membrane. Once there, the SRP-SR complex spontaneously changed its shape and deposited the cargo at the membrane, like a tiny Transformer toy morphing from a semi-truck delivering goods into a forklift that unloads them. The scientists described their discovery in a recent paper in the Proceedings of the National Academy of Sciences.

"The Transformer analogy is very appropriate," says Shan. "The 'truck' is able to sense that cargo has been loaded and starts the engine running without instructions from a driver. It can also sense that it has arrived at the destination and, without workers coming to unload the goods, is able to switch on another system to do that by itself." This self-sufficient system, she says, represents "a new way that biology builds switches to regulate complex cellular pathways."

Shan and colleagues also found that the presence of protein cargo delays the breakdown of a small-molecule energy carrier called guanosine triphosphate, or GTP, from which the SRP and SR harvest the energy to form a complex with each other and to undergo all their molecular transformations. "GTP hydrolysis is like a timer that allows the SRP-SR complex to exist for a specified period of time before turning it off. By delaying this timer, the SRP-SR complex persists about 10 times longer than it would without the cargo. This ensures that there is sufficient time for the cargo to be properly delivered to the membrane," Shan says.

"Understanding which steps are important for protein delivery by the SRP could allow the development of medications that prevent diseases that result from defects in the pathway," Shan says. For example, prion disease can be caused by tiny snippets of misfolded prion proteins that accumulate in the cytoplasm of cells when the SRP pathway does not work properly. The accumulation of cytoplasmic prions leads to the degeneration of neurons, and the eventual death of the affected organism."

The research could also lead to the development of novel artificial delivery systems that can shuttle particular proteins to specific locations, and may spur the design of new types of antibiotics that target the SRP protein in bacteria. Blocking the bacterial SRP will indeed kill bacteria, Shan says, but because humans have SRP proteins, it "will also likely affect the operation of cells in your body. Detailed mechanistic studies are required to figure out the difference between the mammalian and the bacteria SRP pathway, and find places to intervene where the bacterial SRP is uniquely susceptible."

Shan's paper, "Multiple conformational switches in a GTPase complex control co-translational protein targeting," was coauthored by Xin Zhang, a graduate student at Caltech, and Christiane Schaffitzel and Nenad Ban from the Swiss Federal Institute of Technology. The work was funded by the National Institutes of Health and the Swiss National Science Foundation.

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Kathy Svitil
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Caltech 4D Microscope Revolutionizes the Way We Look at the Nano World

PASADENA, Calif.-- More than a century ago, the development of the earliest motion picture technology made what had been previously thought "magical" a reality: capturing and recreating the movement and dynamism of the world around us. A breakthrough technology based on new concepts has now accomplished a similar feat, but on an atomic scale--by allowing, for the first time, the real-time, real-space visualization of fleeting changes in the structure and shape of matter barely a billionth of a meter in size.

Such "movies" of atomic changes in materials of gold and graphite, obtained using the technique, are featured in a paper appearing in the November 21 issue of the journal Science. (4D microscopy videos can be viewed at http://ust.caltech.edu/movie_gallery/.) A patent on the conceptual framework of this approach was granted to the California Institute of Technology (Caltech) in 2006.

The new technique, dubbed four-dimensional (4D) electron microscopy, was developed in the Physical Biology Center for Ultrafast Science and Technology, directed by Ahmed Zewail, the Linus Pauling Professor of Chemistry and professor of physics at Caltech, and winner of the 1999 Nobel Prize in Chemistry.

Zewail was awarded the Nobel Prize for pioneering the science of femtochemistry, the use of ultrashort laser flashes to observe fundamental chemical reactions--atoms uniting into molecules, then breaking apart back into atoms--occurring at the timescale of the femtosecond, or one millionth of a billionth of a second. The work "captured atoms and molecules in motion," Zewail says, akin to the freeze-frame stills snapped by 19th-century photographer Eadweard Muybridge of a galloping horse (which proved for the first time that a horse does indeed lift all four hooves off the ground as it gallops) and other moving objects.

Snapshots of molecules in motion "gave us the time dimension," Zewail says, "but what we didn't have was the dimensions of space, the structure. We didn't know what the horse looked like. Did it have a long tail? Beautiful eyes? My dream since 1999 was to come up with a way to look not just at time but also at the spatial domain; to see the architecture of a complex system at the atomic scale, as it changes over time, be it for physical or biological matter."

Scientists can observe the static structure of objects with a resolution that is better than a billionth of a meter in length using electron microscopes, which generate a stream of individual electrons that scatter off objects to produce an image. Electrons are used to visualize the smallest of objects, on the atomic scale, because the wavelength of the radiation source used by a microscope must be shorter than the space between the atoms. This can be accomplished using electrons, and in particular--because the wavelength of an electron shrinks as its velocity increases--by electrons that have been accelerated to dizzying speeds.

But just having electrons isn't sufficient to capture the behavior of atoms in both space and time; the electrons have to be carefully doled out, so that they arrive at the sample at specific time intervals. Zewail and his colleagues have achieved this by introducing the fourth dimension of time into high-resolution electron microscopy, in what has been termed ultrafast "single-electron" imaging, where every electron trajectory is precisely controlled in time and space.

The resulting image produced by each electron represents a femtosecond still at that moment in time. Like the frames in a film, the sequential images generated by many millions of such images can be assembled into a digital movie of motion at the atomic scale.

As reported in the Science paper, Zewail and colleagues applied their new 4D electron microscopy to observe the behavior of the atoms in superthin sheets of gold and graphite. Graphite, the material in pencils, consists of layers of carbon atoms locked into a sheet-like array. The atoms move in a unique and coherent way on the femtosecond timescale.

However, the researchers found that on a slightly longer, picosecond (one thousandth of a billionth of a second) scale, the graphite nanosheets produce sound waves. In the images, they directly visualized the elastic movements of the sheets and determined the force holding them together, which is described by a stress-strain property known as "Young's modulus." The 4D movies produced from the frames revealed the behavior in space and time.

In a second paper in the current issue of the journal Nano Letters, Zewail and his colleagues described their visualization of the changes in a nanometer-thick graphite membrane on a longer time scale, up to a thousandth of a second. The researchers first blasted the sample with a pulse of heat. The heated carbon atoms began to vibrate in a random, nonsynchronized fashion. Over time, however, the oscillations of the individual atoms became synchronized as different modes of the material locked in phase, emerging to become a heartbeat-like "drumming." Digital video, slowed down more than a billion times, illustrates this nano-drumming mechanical phenomenon, which displays a well-defined resonance that is nearly 100 times higher than can be detected by the human eardrum.

"With this 4D imaging technique, atomic-scale motions, which lead to structural, morphological, and nanomechanical phenomena, can now be visualized directly, and hopefully understood," says Zewail, who is now expanding the research to biological imaging within cells in collaboration with Grant Jensen, an associate professor of biology at Caltech.

The researchers are currently using the 4D microscope to image the components of cells, such as proteins and ribosomes, the cellular machinery that makes proteins. They have already produced images of a stained rat cell and, more recently, of a protein crystal and cell in vitreous water. "The goal is to enhance the structural resolution in the images of these biomaterials by taking single-pulse snapshots before they move or deteriorate, and to follow their dynamics in real time," Zewail says.

In a recent commentary on the development, Sir John Thomas of Cambridge University, who is a world-renowned expert in electron microscopy, said the invention and its applications are "revolutionary." "The door is now open for myriad explorations in the physical and biological sciences," Thomas added.

"The sequences of images produced by this technique are remarkable," says David Tirrell, chair of Caltech's Division of Chemistry and Chemical Engineering. "They not only provide unprecedented insights into molecular and materials behavior--they do so in an especially satisfying fashion by allowing direct observation of complex structural changes in real space and real time. These experiments will lead us to fundamentally new ways of thinking about molecules and materials."

"Advances in imaging, concepts, and technology for visualization are fundamental to progress in diverse scientific and engineering fields," says Edward M. Stolper, Caltech's provost. "Caltech has made a commitment to leadership across the many physical and biological disciplines in which imaging plays a critical role. Ahmed's pioneering work is trailblazing new frontiers of science and technology."

Two centers supported the development of this technology: the Laboratory for Molecular Science, funded by the National Science Foundation, and the Physical Biology Center, funded by Gordon and Betty Moore Foundation. The work was also supported by grants from the Air Force Office of Scientific Research, the National Science Foundation, and the National Institutes of Health.

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Writer: 
Kathy Svitil
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Four Caltech Faculty Members Named Among 100 Chemical Engineers of the Modern Era

American Institute of Chemical Engineers lauds Frances Arnold, Mark Davis, Julia Kornfield, and John Seinfeld

PASADENA, Calif.--Four members of the 11-member chemical engineering faculty at the California Institute of Technology (Caltech) were honored by the American Institute of Chemical Engineers (AIChE) in their list of 100 Chemical Engineers of the Modern Era, published in the October issue of their magazine, Chemical Engineering Progress.

"Now in its second century, the chemical engineering profession--like the Institute--has been shaped and sustained by the achievements, leadership and imagination of thousands of engineers," the AIChE wrote in introducing its list.

The four Caltech chemical engineers named were

* Frances H. Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering and Biochemistry, who was recognized for "research on engineering biological systems, particular proteins and genetic regulatory networks, (e.g., using novel enzymes to catalyze cellulose hydrolysis)." The AIChE also noted that Arnold is the only woman to have been elected to all three branches of the National Academies: the National Academy of Engineering (in 2000), the Institute of Medicine (in 2004), and the National Academy of Sciences (in 2008).

* Mark E. Davis, the Warren and Katharine Schlinger Professor of Chemical Engineering, who was recognized for "pioneering work in new catalytic materials and chemical sensors using ceramics and electronic materials."

* Julia A. Kornfield, professor of chemical engineering, who was recognized for "work on polymer blend dynamics; flow alignment of liquid-crystalline and block polymers; physical aspects of new biomedical materials."

* John H. Seinfeld, the Louis E. Nohl Professor and professor of chemical engineering, who was recognized for "developing first models describing urban air quality" and for being "one of first to describe linkage between urban ozone and global climate change."

"I'm very proud that my colleagues have been recognized in this way by the most important professional organization in the field of chemical engineering. Their selection reflects well on the Caltech approach to things, which is to stay small while maintaining the highest possible standards in education and research," says David Tirrell, chair of Caltech's Division of Chemistry and Chemical Engineering.

"At Caltech, I'm inspired to work on the hardest problems, because the students here can solve them," adds Frances Arnold. "This honor, awarded to almost half our department, certainly recognizes that."

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