Among the first cells of the immune system to respond to microorganisms that invade our body are neutrophils.  Although neutrophils are considered the “good guys” in such circumstances, they also contribute to the noninfectious chronic inflammation that underlies various diseases, including autoimmune diseases such as rheumatoid arthritis.  One mechanism by which neutrophils protect us is to internalize microorganisms and destroy them using proteins known as neutrophil serine proteases (NSPs), but whether NSPs have a role in noninfectious chronic inflammation has not been clearly determined.  However, using mice lacking two very similar NSPs, PR3 and NE, a team of researchers at the Max-Planck-Institute of Neurobiology, Germany, have now shown that these two NSPs have a crucial role in one form of noninfectious chronic inflammation.  Detailed analysis revealed that PR3 and NE destroy an anti-inflammatory molecule known as PGRN and in this way help to promote inflammation in the absence of invading microorganisms.  The authors therefore suggest that these data provide rationale for considering inhibitors of NSPs as anti-inflammatory drugs.

A small molecule that locks an essential enzyme in an inactive form could one day form the basis of a new class of unbeatable, species-specific antibiotics, according to researchers at Fox Chase Cancer Center.

Their findings, highlighted on the cover of the June 23 issue of the journal Chemistry & Biology, take advantage of an emerging body of science regarding “morpheeins” – proteins made from individual components that are capable of spontaneously reconfiguring themselves into different shapes within living cells.

The researchers discovered a small molecule, which they have named morphlock-1, binds the inactive form of a protein known as porphobilinogen synthase (PBGS), an enzyme used by nearly all forms of cellular life.  The functioning form of PBGS is built from eight identical component parts – in what is called an octamer configuration – and is essential among nearly all forms of life in the processes that enable cells to use energy.  The other configuration is made of six parts – or a hexamer configuration – and serves as a “standby” mode for the protein.

“As the name suggests, morphlock-1 essentially locks the hexamer configuration into place, preventing its protein subunits from reconfiguring into the active assembly,” says lead investigator Eileen Jaffe, Ph.D, a Senior Member of Fox Chase.  “Targeting morpheeins in their inactive assemblies provides an entirely new approach to drug discovery.”

While their study was performed using a pea plant-version of PBGS, the researchers have reason to believe the principle could apply to bacterial versions of PBGS as well.  “Using morphlock-1 as a base, we are seeking to fine tune the molecule so that it blocks just the bacterial version of the PBGS enzyme, ” Jaffe says.

“Because PBGS is so crucial for life, the part of the enzyme where chemistry happens is highly conserved through evolution,” Jaffe says, meaning that an all-around PBGS-inhibiting drug would harm bacteria, peas and people alike.  The area where the potential drug binds to the hexamer form of the protein, however, has been found to differ among species, depending how far the organisms have evolved from each other.

When PBGS is in its inactive hexamer form, there is a small cavity on the surface of the assembled complex.  Using computer docking techniques, Jaffe and her Fox Chase colleagues identified a suite of small molecules predicted to bind to this cavity.

The researchers then bought and tested a selection of these molecules in the lab to see if any of them stabilized the pea PBGS in its hexamer assembly.  One inhibitor in particular, given the name morphlock-1, potently drove the formation of the hexamer in pea PBGS, but not in that of humans, fruit flies, or the infectious bacteria Pseudomonas aeruginosa, or Vibrio cholerae, the latter of which causes cholera.  Morphlock-1 is a potent inhibitor of pea PBGS, but not of the PBGS from these other organisms.

Jaffe coined the term “morpheein” in 2005 after a study of the structure of PBGS revealed its shape-shifting tendencies.  While initially met with skepticism because the existence of morpheeins contradicts some classic concepts about protein structure and function, subsequent studies have reinforced that PBGS (and perhaps other proteins) exhibits this behavior.  According to Jaffe, this study is the first to make use of alternate morpheein shapes as a potential strategy for drug discovery, in general, particularly for antibiotics.

“Multi-drug resistance drives the need for developing new antibiotics,” Jaffe says.  “Since drugs that stabilize the inactive PBGS hexamer need not be chemically similar to each other, it will be difficult for the bacterium to develop complete resistance to a cocktail of such compounds.”

A tiny but powerful engine that propels the bacterium Bacillus subtilis through liquids is disengaged from the corkscrew-like flagellum by a protein clutch, Indiana University Bloomington and Harvard University scientists have learned. Their report appears in this week’s Science.Scientists have long known what drives the flagellum to spin, but what causes the flagellum to stop spinning — temporarily or permanently — was unknown.

“We think it’s pretty cool that evolving bacteria and human engineers arrived at a similar solution to the same problem,” said IU Bloomington biologist Daniel Kearns, who led the project. “How do you temporarily stop a motor once it gets going?”

The action of the protein they discovered, EpsE, is very similar to that of a car clutch. In cars, the clutch controls whether a car’s engine is connected to the parts that spin its wheels. With the engine and gears disengaged from each other, the car may continue to move, but only because of its prior momentum; the wheels are no longer powered.

EpsE is thought to “sit down,” as Kearns describes it, on the flagellum’s rotor, a donut-shaped structure at the base of the flagellum. EpsE’s interaction with a rotor protein called FliG causes a shape change in the rotor that disengages it from the flagellum’s proton-powered engine.

The discovery of EpsE and its function was accidental. Kearns and colleagues were actually interested in learning more about the genes that cause individual cells of B. subtilis to cease wandering in solitude and take up residence in a massively communal, stationary assemblage called a biofilm. The stability of biofilms can be jeopardized by hyperactive bacterial cells whose flagella continue to spin.

“We were trying to get at how the bacterium’s ability to move and biofilm formation are balanced,” Kearns said. “We were looking for the genes that affected whether the cells are mobile or stationary. Although B. subtilis is harmless, biofilms are often associated with infections by pathogenic bacteria. Understanding biofilm formation may eventually prove useful in combating bacterial infections.”

Once the scientists learned EpsE was involved in repressing flagellar motion, they devised two possible explanations for how EpsE acts. The first was that EpsE acts like a brake by pushing a non-moving part against a moving part and locking up the works. The other possibility, they imagined, was that EpsE acts like a clutch, disengaging one moving part from another. In this latter scenario, the engine can no longer drive flagellar spinning because key moving parts are no longer in contact. In this case, the flagellum would still have freedom of motion, listless as it might be.

To determine which hypothesis was correct, the scientists decided it best to let the tail wag the dog. They attached the tail end of the flagellum to a glass slide and examined the movement of the entire cell in the presence and absence of EpsE. In the absence of EpsE, the entire cell rotated once every five seconds. In the presence of EpsE, the cells stopped but could rotate passively, pushed by disturbances in the environment (Brownian motion). If EpsE acted like a brake, the cells would not have rotated at all.

The researchers also learned that when the cell begins producing EpsE, it takes about 15 minutes before the flagellar machinery is disabled.

“This makes a lot of sense as far as the cell is concerned,” Kearns said. “The flagellum is a giant, very expensive structure. Often when a cell no longer needs something, it might destroy it and recycle the parts. But here, because the flagellum is so big and complex, doing that is not very cost effective. We think the clutch prevents the flagellum from rotating when constrained by the sticky matrix of the biofilm.”

The discovery may give nanotechnologists ideas about how to regulate tiny engines of their own creation. The flagellum is one of nature’s smallest and most powerful motors — ones like those produced by B. subtilis can rotate more than 200 times per second, driven by 1,400 piconewton-nanometers of torque. That’s quite a bit of (miniature) horsepower for a machine whose width stretches only a few dozen nanometers.

Against currently held dogma, scientists at the Universities of Cambridge and Bristol have revealed that the interactions within membrane complexes can be maintained intact in the vacuum of a mass spectrometer. Their research is published in this week’s edition of Science Express.

The researchers were surprised to discover that membrane complexes could remain associated as it has always been assumed that they would not survive once transferred to the alien conditions inside the mass spectrometer.

“Even if interactions between proteins within the membrane could be maintained we would not have expected them to remain associated with proteins in the cell’s interior,” says Carol Robinson, Principal Investigator and Royal Society Research Professor at the University of Cambridge’s Department of Chemistry.

Cellular membranes surround cells and provide the ultimate in cellular security; nothing can get into a cell without the say so of membrane proteins – the worker molecules that reside in the membrane wall and provide tightly regulated entry points. This natural home of membrane proteins excludes water, yet methods available to study proteins at high resolution revolve round aqueous environments. The ability to “fly” intact membrane proteins in a mass spectrometer paves the way for weighing the proteins and identifying the molecular partners they work with in nature.

The new research, funded by the Biotechnology and Biological Sciences Research Council, will enable scientists to investigate membrane complexes with from a variety of sources and with a range of small molecules. Since about 60% of all drug targets are membrane proteins this is a significant discovery.

Ever since Professor Robinson first flew soluble protein complexes in a mass spectrometer in 1996, she has wanted to do the same with membrane complexes. Collaborating with a membrane biochemistry group in Bristol, led by Professor Paula Booth, she began to think of ways of studying these most challenging assemblies.

Dr Nelson Barrera a post-doctoral researcher in Chile, though experienced in membrane biochemistry, was a new recruit to mass spectrometry. He was largely unaware of the difficulties that had previously been encountered and approached the problem in a new way. Rather than trying to remove the detergent (used to keep the protein intact in solution once outside the natural membrane) he maintained the detergent in unusually high amounts. He then deliberately destroyed this protective detergent layer once in the gas phase. This allowed him to liberate the intact assembly. He was also able to remove units from the modular assembly in the gas phase, just as in solution.

Professor Robinson adds: “I am very excited by this finding given the importance of membrane complexes in guarding the entrance and exit to cells. The type of proteins we have been studying, for example, are involved in drug resistance in cancer cells and antibiotic resistance of bacteria.

“I look forward to exploiting this discovery to the full; not only in characterising the many membrane complexes for which controversy exists but also in discovering new assemblies and in investigating the potential of this approach in drug discovery.”

Professor Paula Booth, at the University of Bristol added: “This is a major advance that helps us understand how nature constructs cellular life. The membrane wall of cells is a precision-made, complex and highly regulated structure. We are now much better equipped to understand this incredible, natural self-assembly feat.”

Coronaviruses, a group including the well-known SARS virus, are the causative agents of many respiratory and enteric infections in humans and animals. As with all viruses, virtually every step of their infection cycle depends on host cellular factors. As the first, most crucial step after their penetration into cells, coronaviruses assemble huge RNA replication “factory” complexes in association with characteristic, newly induced double membrane vesicles. The cellular pathways hijacked by these plus-strand RNA viruses to create these “factories” have thus far not been elucidated.

The researchers, led by Cornelis A. M. de Haan, showed that RNA replication of mouse hepatitis coronavirus (MHV) was inhibited by a drug — brefeldin A — that disrupts the central station in the cell’s secretory pathway, the Golgi complex. Consistently, depletion of both the cellular target of brefeldin A, a factor called GBF1, and its downstream target, ARF1, was also shown to negatively affect coronavirus infection.

The researchers conclude that “an intimate association exists between the early secretory pathway and MHV replication.” They speculate that, while GBF1 and ARF1 are not involved in the formation of the viral replication structures, they probably play a key role in their maturation or functioning. As this work was limited to the mouse hepatitis coronavirus, an interesting next step would be to study the importance of GBF1 and ARF1 in the replication of other coronaviruses.

Uncontrolled blood vessel growth is a key feature of many pathological conditions, including the degenerative diabetic eye disease known as diabetic retinopathy.  Understanding the factors involved in the process is vital to developing treatments for the disease.  In a new study, a team of researchers at Keio University, Japan, has revealed a role for the protein LIF in blood vessel growth in mice.

Specifically, mice lacking LIF were observed to have increased blood vessel growth in many regions of the body, but as this study was focused on the eye, the authors homed in on the increased blood vessel growth in the retina of the eye.  Further analysis showed that mice lacking LIF developed more aberrant blood vessels in a model of retinopathy.  Mechanistically, LIF was found to inhibit the proliferation of brain cells known as astrocytes as well as inhibit their production of a factor known to promote blood vessel growth, VEGF.  It therefore seems that LIF is an important part of the communication between tissues and developing blood vessels, meaning that LIF and the signaling pathway it triggers might serve as a target for new treatment approaches for preventing diabetic retinopathy and other diseases that are associated with uncontrolled blood vessel growth, such as cancer.

Montana State University scientists in the Department of Chemistry and Biochemistry published new research this week that could one day affect the lives of millions around the world who suffer from blood iron disorders.
Martin Lawrence (left) and Anoop Sendamarai (MSU photo by Kelly Gorham)
The paper, which will appear in the Proceedings of the National Academy of Sciences, details the work of Associate Professor Martin Lawrence and doctoral candidate Anoop Sendamarai. The pair have spent the past two years studying Steap3, a protein involved in regulating the body’s absorption of iron.

The results of their studies - the first three-dimensional maps of the atoms that make up Steap3 - could allow pharmaceutical companies to someday design drugs to regulate iron levels in the blood.

“Iron is essential,” Lawrence said. “You can’t live without it, but it’s a double-edged sword. Too much of a good thing can kill you.”

Iron serves several important functions in the bloodstream. It carries oxygen, transports electrons within cells and plays an important role in enzyme systems.

Iron irregularities are some of the most common blood disorders in the world. According to the World Health Organization, iron deficiency, which can lead to anemia, affects more than a billion people around the world and can cause developmental and immune system problems.

Conversely, having too much iron, a condition called hemochromatosis, can also hurt the body by releasing destructive free radicals, Lawrence said. Hemochromatosis affects about one in every 300 people and is most common in people of northern European ancestry. Left untreated, it can lead to early death, often by age 50.

“We’re struck by how many people have too much or too little iron,” Lawrence said.

To understand Steap3’s role in transporting and maintaining balanced levels of iron, Lawrence and Sendamarai first had find and purify samples of the protein and then turn those samples into crystals.

Lawrence said the result of the crystallization process, if done correctly, is analogous to the rigid structure of a brick wall. If done incorrectly, it more closely resembles a pile of bricks.

“It’s kind of a black art really more than a science,” Lawrence said. “You can’t always predict the kind of witch’s brew that needs to be around to get it to crystallize.”

He said only a handful of labs in the country are crystallizing iron transport proteins like Steap3, a fact that places MSU on the same shelf as places like Harvard Medical School.

Once crystallized, the samples are shot with a powerful X-ray beam. Electrons in the sample diffract the X-rays, creating patterns on a digital sensor. The technique, called X-ray crystallography, has been used since the 1950s to de-termine the structure of different substances.

In their basement lab in the campus’s New Chemistry Building, Lawrence and Sendamarai then examined the diffraction patterns created by Steap3.

“It’s kind of like a contour map,” Sendamarai said. “Whenever we see the peaks, we know there are atoms.”

Working backward, they can mathematically determine the position of atoms in the protein and display them in three dimensions.

The computer-drawn result, a three-dimensional image that resembles tangled ribbons and strings, is an picture of what the atoms of Steap3 look like.

Sendamarai said having that picture, which depicts all the nooks and crannies on the protein’s surface, could allow drug companies to design drugs to fit those spots like puzzle pieces.

If a future drug fits those nooks just right, it could help treat hemochromatosis. From there, Sendamarai said it would be conceivable to work backward and possibly treat iron deficiencies or anemia.

Lawrence said that Steap3 is only one in a family of proteins that affect iron transport. This summer, in addition to continuing to study Steap3, Lawrence and Sendamarai hope to learn whether the lab will receive a grant from the National Institutes of Health to work on other iron transport proteins.

“It’s a critical step towards toward learning to modulate iron levels in patients with too much or too little iron,” Sendamarai said. “But, there are a lot of question marks left in iron transport. It’s a big field.”

Biologists from Austria and Singapore developed a technique that adds a new twist on the relationship between biology and art. In an article recently published online in The FASEB Journal (http://www.fasebj.org) and scheduled for the August 2008 print issue, these researchers describe how they were able to coat—or paint—viruses with proteins. This breakthrough should give a much-needed boost to the efficiency of some forms of gene therapy, help track and treat viral disease and evolution, improve the efficiency of vaccines, and ultimately allow health care professionals track the movement of viral infections within the body. Specifically, the new method should make it easier to track and treat infectious diseases such as HIV/AIDS, influenza, hepatitis C, and dengue fever. And because viruses can also be used to introduce biotechnology drugs and replacement genes, and act as vaccines, this research should lead to new treatments for cancer, cardiovascular, metabolic and inherited disorders.

“This technology should provide a new tool for the treatment of many diseases,” said Brian Salmons, one of scientists who co-authored the study. “Even if you are working with a virus that is unknown or poorly characterized, it is still possible to modify or paint it. This is very interesting for emerging diseases.”

In the article, Salmons and colleagues explain how they mixed purified proteins (glycosylphophatidylinositol anchor proteins) with lipid membranes to make it possible to bind these proteins to the outer “skin” (the lipid envelope) of viruses. Even with the new paint job, the viruses remained infectious. While the experiment only involved one type of protein and two types of viral vectors, Salmons says the technique could be expanded and used to apply “paint” made up of other proteins, dyes, and a variety of unique markers.

“Biology and art converge daily: people paint their nails, color their hair, and tattoo their skin,” said Gerald Weissmann, M.D., Editor-in-Chief of The FASEB Journal. “Now this convergence has entered a new dimension as painted viruses permit scientists to track, cure and prevent disease.”

Amyloid deposits in tissues and organs are linked to a number of diseases, including Alzheimer’s, Parkinson’s, type II diabetes, and prion diseases such as BSE. However, amyloids are not just pathological substances; they have potential as a nanomaterials. “The potential applications of these supramolecular assemblies exceed those of synthetic polymers,” state Ehud Gazit and co-author Izhack Cherny in the journal Angewandte Chemie, “since the building blocks may introduce biological function in addition to mechanical properties.”

© Wiley-VCH

Even in nature, amyloids are not merely abnormal, incorrectly folded proteins; they are physiological components of organisms. For example, they are an important protective material in the egg envelopes of insects and fish. They are also involved in the formation of the biofilms of many bacteria, a coating on the surface of the bacterial cells that protects them from antimicrobial substances and facilitates their attachment to surfaces.

Amyloid fibrils are bundles of highly ordered protein filaments made of ladder-like strands and can be several micrometers long. In cross-section, amyloids appear as hollow cylinders or ribbons. Although amyloid fibrils are proteins, they more closely resemble synthetic polymers (plastics) than the usual globular proteins. Amyloids can display amazing mechanical properties similar to spider silk. Spider silk is, by weight, significantly stronger than steel and can be stretched to many times its original length without tearing— properties that have not been reproducible with synthetic fibers.

“The self-assembly properties of amyloids, together with their observed plasticity, makes them attractive natural building blocks for the design of new nanostructures and nanomaterials,” according to the authors from the University of Tel Aviv (Israel). “These building blocks can be broadly varied by means of simple molecular biological techniques.” Surfaces could be given tailored and biocompatible coatings, for example, in analytical flow devices for medical technology or bioanalysis. Other ideas include amyloid hydrogels for the encapsulation and controlled release of drugs and for scaffolds for three-dimensional cell cultures and tissue engineering. Functional proteins such as enzymes could be bound to amyloid-forming sequences to mimic biological processes.

Amyloid fibrils are also suitable as matrices for nanostructures. For example, it has been possible to produce a conducting nanoscale coaxial cable by filling amyloid nanotubes with sliver and externally coating them with gold.

A team of researchers at the National Institute of Standards and Technology (NIST) have demonstrated a new device that creates nanodroplet “test tubes” for studying individual proteins under conditions that mimic the crowded confines of a living cell. “By confining individual proteins in nanodroplets of water, researchers can directly observe the dynamics and structural changes of these biomolecules,” says physicist Lori Goldner, a coauthor of the paper* published in Langmuir.

With the flip of a switch: Nanodrop “test tubes” are created by an electronic switch that causes a micropipette to jerk back and leave behind a droplet less that 1 micron in diameter for study.
Credit: NIST

Researchers recently have turned their attention to the role that crowding plays in the behavior of proteins and other biomolecules—there is not much extra space in a cell. NIST’s nanodroplets can mimic the crowded environment in cells where the proteins live while providing advantages over other techniques to confine or immobilize proteins for study that may interfere with or damage the protein. This more realistic setting can help researchers study the molecular basis of disease and supply information for developing new pharmaceuticals. For example, misfolded proteins play a role in many illnesses including Type 2 diabetes, Alzheimer’s and Parkinson’s diseases. By seeing how proteins fold in these nanodroplets, researchers may gain new insight into these ailments and may find new therapies.

The NIST nanodroplet delivery system uses tiny glass micropipettes to create tiny water droplets suspended in an oily fluid for study under a microscope. An applied pressure forces the water solution containing protein test subjects to the tip of the micropipette as it sits immersed in a small drop of oil on the microscope stage. Then, like a magician whipping a tablecloth off a table while leaving the dinnerware behind, an electronic switch causes the pipette to jerk back, leaving behind a small droplet typically less than a micrometer in diameter.

The droplet is held in place with a laser “optical tweezer,” and another laser is used to excite fluorescence from the molecule or molecules in the droplet. In one set of fluorescence experiments, explains Goldner, “The molecules seem unperturbed by their confinement—they do not stick to the walls or leave the container—important facts to know for doing nanochemistry or single-molecule biophysics.” Similar to a previous work (see “‘Micro-boxes’ of Water Used to Study Single Molecules”, Tech Beat July 20, 2006), researchers also demonstrated that single fluorescent protein molecules could be detected inside the droplets.

Fluorescence can reveal the number of molecules within the nanodroplet and can show the motion or structural changes of the confined molecule or molecules, allowing researchers to study how two or more proteins interact. By using only a few molecules and tiny amounts of reagents, the technique also minimizes the need for expensive or toxic chemicals.

Reference:

* J. Tang, A.M. Jofre, G.M. Lowman, R.B. Kishore, J.E. Reiner, K. Helmerson, L.S. Goldner and M.E. Greene. Green fluorescent protein in inertially injected aqueous nanodroplets. Published in Langmuir, ASAP Article, Web release date: March 27, 2008.

Shown is an image of bacteriophage Epsilon15 studied by Wen Jiang, an assistant professor of biological sciences at Purdue. The bacteriophage is shown at a resolution of 4.5 angstrom — the highest resolution achieved for a living organism of this size. Credit Graphic/Wen Jiang lab

WEST LAFAYETTE, Ind. - A team led by a Purdue University researcher has achieved images of a virus in detail two times greater than had previously been achieved.

Wen Jiang, an assistant professor of biological sciences at Purdue, led a research team that used the emerging technique of single-particle electron cryomicroscopy to capture a three-dimensional image of a virus at a resolution of 4.5 angstroms. Approximately 1 million angstroms would equal the diameter of a human hair.

“This is one of the first projects to refine the technique to the point of near atomic-level resolution,” said Jiang, who also is a member of Purdue’s structural biology group. “This breaks a threshold and allows us to now see a whole new level of detail in the structure. This is the highest resolution ever achieved for a living organism of this size.”

Details of the structure of a virus provide valuable information for development of disease treatments, he said.

“If we understand the system - how the virus particles assemble and how they infect a host cell - it will greatly improve our ability to design a treatment,” Jiang said. “Structural biologists perform the basic science and provide information to help those working on the clinical aspects.”

A paper detailing the work was published in the Feb. 28 issue of Nature.

Roger Hendrix, a professor of biological sciences at the University of Pittsburgh, said what is learned about viruses can be applied to many other biological systems.

“Understanding the proteins that create the structure of a virus gives us insight into the tiny biological machines found throughout our bodies,” he said. “Getting to 4.5 angstrom using this technique is a watershed of sorts because it is the first time we can actually trace the polypeptide chain - the backbone of proteins. Now we can see the tiny gears and levers that allow the proteins to move and interact as they carry out their intricate biological roles.”

The imaging technique, called cryo-EM, has the added benefit of maintaining the sample being studied in a state very similar to its natural environment. Other imaging techniques used regularly, such as X-ray crystallography, require the sample be manipulated.

“This method offers a new approach for modeling the structure of proteins in other macromolecular assemblies, such as DNA, at near-native states,” Jiang said. “The sample is purified in a solution that is very similar to the environment that would be found in a host cell. It is as if the virus is frozen in glass and it is alive and infectious while we examine it.”

In addition to Jiang, Matthew L. Baker, Joanita Jakana and Wah Chiu from Baylor College of Medicine, and Peter R. Weigele and Jonathan King from Massachusetts Institute of Technology worked on the project, which was funded by the National Institutes of Health and the National Science Foundation.

The team obtained a three-dimensional map of the capsid, or protein shell, of the epsilon15 bacteriophage, a virus that infects bacteria and is a member of a family of viruses that are the most abundant life forms on Earth, Jiang said.

Other methods of determining the structure could not be used for this family of virus. None had been successfully crystallized, and the complexity of members of this family had prevented evaluation through the genome sequence alone.

“This demonstration shows that cryo-EM is doable and is a major step in reaching the full potential of this technique,” he said. “The goal is to have it reach a 3 to 4 angstrom resolution, which would allow us to clearly see the amino acids that make up a protein.”

In electron microscopy, a beam of electrons takes the place of the light beam used in a conventional microscope. The use of electrons instead of light allows the microscope to “see” in much greater detail.

Cryo-EM cools specimens to temperatures well below the freezing point of water. This decreases damage from the electron beam and allows the specimens to be examined for a longer period of time. Longer exposure time allows for sharper, more detailed images.

Researchers using cryo-EM had obtained images at a resolution of 6-9 angstroms but could not differentiate between smaller elements of the structure spaced only 4.5 angstroms apart.

“There are different elements that make up the protein building blocks of the virus,” Jiang said. “It is like examining a striped blanket. From a distance, the stripes blur together and the blanket appears to be one solid color. As you get closer you can see the different stripes, and if you use a magnifying glass you can see the strands of string that make up the material. The resolution needs to be smaller than the distance between the strands of thread in order to see two separate strands.

“By being able to zoom in, researchers were able to see components that blurred together at the earlier achieved resolution.”

Cryo-EM requires high-end electron microscopes and powerful computing resources. The research team used the Baylor College of Medicine’s cryoelectron microscope. It is expected that Purdue will install a state-of-the-art cryoelectron microscope in 2009.

In 2006 Purdue received a $2 million grant from the National Institute of Health to purchase the microscope. It will be installed in Hockmeyer Hall of Structural Biology, expected to open in 2009.

Computer programs are used to extract the signal from the microscope and to combine thousands of two-dimensional images into an accurate three-dimensional image that maps the structure of the virus. This requires use of a large data set and could not have been done without the resources of Purdue’s Office of Information Technology, or ItaP, Jiang said.

Jiang used Purdue’s Condor program - which links computers including desktop machines and large, powerful research computers - to create the largest distributed computing network at a university.

“ITaP provided us with computational power at the supercomputer scale that was necessary for this work,” he said. “Purdue’s Condor program allowed us to take advantage of the power of 7,000 computers. This was a critical element to our success.”

Jiang plans to continue to refine every step of the process to improve the capabilities of the technique and to examine more medically relevant virus species.

Purdue’s structural biology group studies a diverse group of problems, including cellular signaling pathways, RNA catalysis, bioremediation, molecular evolution, viral entry, viral replication and viral pathogenesis. Researchers use a combination of X-ray crystallography, electron cryomicroscopy, NMR spectroscopy, and advanced computational and modeling tools to study these problems.

Illinois postdoctoral researcher Feng-Jie Sun (left) and crop sciences professor Gustavo Caetano-Anollés began with the idea that understanding the structural properties of tRNA would shed light on how organisms and viruses evolved. Photo by L. Brian Stauffer, U. of I. News Bureau.

Transfer RNA (also called tRNA) is an ancient molecule, central to every task a cell performs and thus essential to all life. A new study from the University of Illinois indicates that it is also a great historian, preserving some of the earliest and most profound events of the evolutionary past in its structure.

The study, co-written by Gustavo Caetano-Anollés, a professor of crop sciences, and postdoctoral researcher Feng-Jie Sun, appears March 7 in PLoS Computational Biology. Caetano-Anollés is an affiliate of the U. of I. Institute for Genomic Biology.

Of the thousands of RNAs so far identified, transfer RNA (tRNA) is the most direct intermediary between genes and proteins. Like many other RNAs (ribonucleic acids), tRNA aids in translating genes into the chains of amino acids that make up proteins. With the help of a highly targeted enzyme, each tRNA molecule recognizes and latches onto a specific amino acid, which it carries into the protein-building machinery. In order to successfully add its amino acid to the end of a growing protein, tRNA must also accurately read a coded segment of messenger RNA, which gives instructions for the exact sequence of amino acids in the protein.

The fact that tRNA is so central to the task of building proteins probably means that it has been around for a long time, Caetano-Anollés said. His inquiry began with a hunch that understanding the structural properties of tRNA would shed light on how organisms and viruses evolved.

“Perhaps in evolution there are things that are so fundamental that they are kept, held onto, for millions or even billions of years,” Caetano-Anollés said. “Those are the fossils, the molecular fossils, that tell us about the past. Therefore, studying these molecules can address fundamental questions in biology and evolution.”

All tRNAs assemble themselves into a shape that, if flattened, resembles a cloverleaf. Patterns in these structures give clues to early evolutionary history. The red areas of the molecule pictured above are the most ancient. Image courtesy of Gustavo Caetano-Anollés.

All tRNAs assemble themselves into a shape that, if flattened, resembles a cloverleaf. The team began by looking for patterns in this cloverleaf structure, using detailed data from hundreds of molecules representing viruses and each of the three superkingdoms of life: archaea, bacteria and eukarya.

The researchers converted all distinguishing features of the individual tRNA cloverleaf structures into coded characters, a process that allowed a computerized search for the most “parsimonious” (that is, the simplest, most probable) tRNA family tree. They conducted the same analysis on the tRNAs of each of the superkingdoms, to see how far these groupings diverged from the overall tree. This comparison allowed them to determine the order in which viruses and each of the superkingdoms diverged.

The new analysis supports an earlier study that suggested that the archaea were the first to arise as an evolutionarily distinguishable group. Archaea are microbes that can survive in boiling acid, near sulfurous ocean vents or in other extreme environments. The earlier study, also led by Caetano-Anollés, analyzed the vast catalog of protein folds – those precisely configured regions in proteins that give them their functionality – as a guidebook to evolutionary history.

“The transfer RNA data matches our earlier data,” Caetano-Anollés said. “This is important because two lines of independent evidence are supporting each other.”

The new analysis also indicates that viruses emerged not long after the archaea, with the superkingdoms eukarya and bacteria following much later – and in that order. This finding may influence the ongoing debate over whether viruses existed prior to, or after, the emergence of living cells, Caetano-Anollés said.

“This supports the idea that viruses arose from the cellular domain,” he said.