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.