New inhibitors of a cancer-causing protein may lead to targeted therapeutics

The activity of protein kinases, a large class of signaling molecules, must be closely regulated or signaling chaos arises within cells. Signaling chaos sets off a process that is implicated in the development of cancers, including solid tumors. Because protein kinases have a central role in cell signaling, researchers have devoted decades of investigation to developing protein kinase inhibitors.

One kinase inhibitor, a drug called Imatinib (or Gleevec), illustrates the profound potential of kinase inhibitors in cancer treatment. The hyperactivity of the Abl kinase is the most common cause of chronic myelogenic leukemia (CML). Imatinab is routinely used to treat CML. Since Imatinib’s Food and Drug Administration-approval in 2001, deaths related to CML have dropped significantly.

Src kinase inhibitors have been used in clinical trials as experimental treatments for many types of solid tumors. However, in contrast to the success of Imatinib for treating CML, a drug based on inhibiting the Src kinase has not been effective in treating solid tumors.

“Think of kinases as a traffic light system that is regulated to keep traffic flowing properly,” says Dr. Seeliger. “But if all lights on a road system are green, chaos occurs. The Src kinase is a dangerous ‘traffic light’ when it stays green. The challenge is to inhibit Src kinase, in other words make it a ‘red light,’ without stopping other Src family of kinases from staying ‘green’ to keep the traffic moving safely.”

In “Highly specific, bisubstrate-competitive Src inhibitors from DNA-templated macrocycles,” the researchers successfully completed two steps that are necessary for the development of new Src kinase-targeted drugs.

First, under the direction of David Liu, Ph.D., Professor, Department of Chemistry, Harvard University, the team developed chemical inhibitors of Src kinase. Dr. Seeliger’s laboratory at Stony Brook then determined the three-dimensional structure of these inhibitors bound to the Src kinase. This second step enabled the team to explain why the inhibitors work to stop the Src kinase but not the other kinases in cultured mammalian cells.

“Using this method, the precise molecular basis of the inhibitory mechanism and Src kinase are revealed,” says Dr. Seeliger. “These results provide new insights into the development of Src-specific inhibitors with potential therapeutic relevance.”

Provided by Stony Brook University (news : web)

Protein 'jailbreak' helps breast cancer cells live

All four proteins were already under suspicion. Researchers, for example, have already tried to assess what levels of HDAC6 in patients with estrogen-receptor positive breast cancer may mean for their prognosis. The results have been inconclusive. The new research suggests that measuring overall levels may not be enough, said the study's senior author Dr. Rachel Altura, associate professor of pediatrics in The Warren Alpert Medical School of Brown University and a pediatric oncologist at Hasbro Children's Hospital.

"We need to look not only at the levels, but also where is it in the cell," she said.

Altura's emphasis on location comes from what her research team found as they tracked and tweaked the comings and goings of survivin in cells. Inside the nucleus, survivin is no problem. Outside the nucleus, but within the cell, it can prevent normal cell death, allowing cancer cells to persist.

In previous work, Altura and her collaborators established that under normal circumstances, CBP chemically regulates survivin, a process called acetylation, and keeps it in the nucleus. The question in the new work was how survivin gets out.

In a series of experiments, what they observed was that in human and mouse breast cancer cells, HDAC6 gathers at the boundary between the nucleus and the rest of the cell, becomes activated by CBP, then binds survivin and undoes its acetylation. This deacetylation allows survivin to then be shuttled out of the nucleus by CRM1.

In the classic jailbreak, CBP is a corrupt guard who looks the other way as HDAC6, the shovel, is smuggled in. The final accomplice, CRM1, is the tunnel with a getaway car on the other end.

Working the new leads

Altura said the research suggests a clear strategy — to keep survivin in the nucleus — and two leads to pursue it, both of which she has already begun working on with collaborators in academia and in the pharmaceutical industry.

One idea is to inhibit HDAC6 in an attempt to prevent it from misregulating the acetylation of survivin. While general HDAC inhibitors are in clinical trials, Altura is optimistic that blocking just HDAC6, using specific inhibitors developed by a colleague in Japan, would have fewer complications.

"You always have to worry about all the things you don't know that you are targeting," she said. "If we can target HDAC6, we can maybe block survivin from coming out of the nucleus and maintain it in its good state."

The other strategy is to block CRM1, Altura said, an idea she is pursuing with a pharmaceutical company in breast cancer cells in the lab. She said preliminary experiments look promising in keeping survivin inside the nucleus and making cancer cells more susceptible to dying.

Provided by Brown University (news : web)

Infrared spectroscopy allows scientists to analyze protein structure on ultrafast timescale

Now, MIT researchers have developed a way to analyze proteins that doesn’t require any pre-treatment. The technique is also extremely fast, allowing scientists to see, for the first time, how a protein changes its shape over picoseconds, or trillionths of a second.

The researchers, led by chemistry professor Andrei Tokmakoff and postdoc Carlos Baiz, describe their new technique this month in the journal Analyst. Their approach builds on a technology known as two-dimensional infrared spectroscopy, which works by shining pulses of infrared light on a molecule and measuring the resulting molecular vibrations. In the new paper, the researchers came up with a way to analyze that data and correlate it with common structural elements found in proteins.

Once assembled, proteins tend to fold into one of two secondary structures, known as alpha helices and beta pleated sheets. In this study, the researchers distinguished between those two structures by examining how bonds between carbon and oxygen — found in each of the amino acids that make up proteins — vibrate when exposed to infrared light. 

In an alpha helix, the carbon-oxygen bonds run parallel to the protein’s backbone; in a beta sheet, those bonds are perpendicular to the sheet. Because of that difference, the bonds vibrate at different frequencies when struck with infrared light. This allows the researchers to calculate the percentage of the amino acids that belong to a helical structure and the percentage that form a beta sheet.

The researchers confirmed the accuracy of their calculations by analyzing a set of proteins whose structures are already known. Their method does not currently reveal the exact structure of a protein, but the researchers are working on ways to determine the arrangements of the sheets and helices from the spectroscopic data.

“In principle, the full structure of the protein is represented in the spectrum. The trick is how to get out the information,” says Baiz, lead author of the paper.

One way to do that is to analyze data from a broader range of infrared wavelengths. The researchers are also developing methods to get information about other bonds within the amino acids.

Because the new method can be performed over millionths of a second, it can be used to study how proteins fold and unfold when denatured by heat. After hitting a protein with a laser blast to heat it up, the researchers can capture a series of snapshots of how the protein unfolds over this very short time period.

“This is the first method that will allow us to take snapshots of the structure of the protein as it’s denatured,” Baiz says. “Usually the way people look at proteins is they start with the unfolded state and they end up with the folded state, so you have two static structures. What we can do now is look at all the structures along the pathway.”

Munira Khalil, an assistant professor of chemistry at the University of Washington, says the ability to track structural changes over time is the technique’s biggest strength. “One big question is how do proteins fold — at what point does it go from a completely disordered structure to an ordered structure?” says Khalil, who was not involved in this research.

This would be particularly useful for studying proteins that cause disease when misfolded, such as the tau protein found in patients with Alzheimer’s disease and the prion that causes Creutzfeldt-Jakob disease.

The method can also measure the structural changes that occur as proteins bind to each other. “If the protein is like a rock, and doesn’t change, then it’s never really going to bind its target or do anything. Those are the types of processes we can look at — the conformational changes that drive biological function,” Baiz says.

Provided by Massachusetts Institute of Technology (news : web)

This story is republished courtesy of MIT News (http://web.mit.edu/newsoffice/), a popular site that covers news about MIT research, innovation and teaching.

Protein behavior might hold the key to synthetic silk

A trans-Atlantic collaboration of scientists has revealed the structure of a key protein of silk and discovered a previously unknown behavior of this protein: to self-organize into tiny fibrils a single molecule in diameter. This discovery sets the stage for the eventual creation of synthetic silk—not just the luxury fabric that’s a product of silkworms, but also the manufacture of ultra-tough spider silk familiar to fans of the Marvel superhero Spider-Man.

Hannes Schniepp cautions that the world’s textile mills aren’t likely to start producing “spidey silk” in the near future, but says his work describing the structure of the silk protein and its self-organizing behavior is an important step in that direction. Schniepp is an assistant professor in the Department of Applied Science at the College of William & Mary. Along with graduate student Minzhen Cai and a set of collaborators at the University of Oxford in the United Kingdom, he has published a paper describing silk at the molecular level.

“Silk is a polymer,” Schniepp explained. “It’s not a synthetically-made polymer, but it’s a polymer made out of proteins.”

Synthetic plastics are polymers, but these macromolecules are common in the natural world, too. Schniepp pointed out that much of the human body—including DNA—is constructed of various polymers. 

“What’s so fascinating about silk is that in terms of its mechanical properties, silk is better than any polymer that we can make synthetically,” he said. “Particularly, certain spider silks are even tougher than Kevlar, the best high-performance polymer we have.”

You can’t farm spiders

For millennia, people have been using the cocoons of silkworms to weave silk cloth. Humans have used spider silk to a much lesser degree, but spiders have proven to be impossible to cultivate: “They start eating each other,” Schniepp says.

Figuring out a process to make synthetic silk has been a sort of Holy Grail of materials science for nearly as long as people have been making silk. After years of scientific study, the exact natures of both the biochemistry and the mechanics of silk creation by silkworms and spiders remain elusive.

“The big question really is how does the spider do it? How does the silkworm do it?,” Schniepp says. “The problem is it’s a tiny animal and it happens really in very small dimensions inside the animal, and it’s really almost impossible to watch what’s going on there.”

He said that most of the scientific study on structure of silk has focused on examination of the product through microscopy and other analytical tools. The study has yielded a fair amount of understanding about the structural nature of silk, but scientists had no idea of what shape an individual silk protein had.

Schniepp and his team at William & Mary took a different approach than most materials scientists, sampling “silk dope,” the gel-like material inside the silkworm that the worm exudes to spin its cocoon.

“A lot of these biomolecules, they’re very sensitive to changes. So the closer you can be to the native state, the more valuable this information is that you get,” he said.

Working with silk dope

Schniepp and his research group examined the silk dope in their McGlothlin-Street Hall laboratory, using an atomic force microscope (AFM), an instrument capable of looking at materials at the nanoscale. Before placing them in the AFM, they prepared their silk dope, diluting the samples with a bit of water, then spun the sample on a plate, so that the silk spread out on the surface.

“When you spin liquid on a plate like this, you shear it. And that does something to these proteins that’s similar to the way that the animals do,” he explained. “They have a gland that produces this material and at the end is something like a nozzle. So they squeeze this material out through the nozzle. To create a similar effect, you shear the solution. By spinning it very quickly, the liquid is forced away, and it is similar to what happens when the animal pushes the silk out.”

A number of curious things happen when the material is sheared. For one thing, the water-soluble silk dope has been transformed into something waterproof. More importantly, the shearing somehow induces individual proteins to “find each other,” as Schniepp describes, and to self-organize into fibrils. One molecule thick, the fibrils are the thinnest possible threads of silk and are precursors to silk fibers.

Seen through AFM magnification, each fibril shows where the individual proteins have conglomerated. The magnification resembles a string of pearls. It’s the first time that the structure of the native silkworm protein has been imaged at such high resolution.

The work on silk is supported by the Jeffress Memorial Trust. Schniepp published his findings in a paper, “Shear-Induced Self-Assembly of Native Silk Proteins into Fibrils Studied by Atomic Force Microscopy” in the journal Biomacromolecules. Fritz Vollrath of Oxford University is a co-author, as is Cai. They are continuing their work on the structure of the material.

“We don’t know what other secrets silk has hidden for us,” Schniepp says.

Provided by The College of William & Mary

Semi-automated 'pathwalking' to build a protein model

In a report that appears online in the journal Structure, the BCM team describes the development of the semi-automated protocol that enables researchers to "rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models."

Taking the 3-D images generated through the process of electron cryo-microscopy and X-ray crystallography, the team developed this computational approach to produce these first-generation models of the proteins' structure or fold without prior knowledge of the protein's sequence or other information.

"This is important in working with big complexes made up of 10 to 30 proteins," said Dr. Matthew Baker, instructor in biochemistry and molecular biology at BCM and the paper's corresponding author. "You might know the structure of one or two proteins, but you want to know how all of those proteins interact with each other. As long as you can separate one protein from another, you can use this technique to make a model of each of the proteins in the complex."

"We borrowed from a classic computer science problem called the 'traveling salesman problem,'" said Dr. Mariah Baker, the paper's first author and a postdoctoral fellow at BCM. "It is in effect a connect-the-dots puzzle without the numbers."

In the traveling salesman problem, computer programmers are asked to figure the best route for a salesman who wants to visits all the cities where he sells just once while minimizing the distance traveled. Pathwalking solves a similar problem for proteins by looking for the optimal path through a 3-D image that connects C-alpha atoms, rather than cities, to form the protein's structure.

The tool is the answer to the dilemma presented by the near-atomic structures that are in the "middle" – not of the highest resolution or the lowest resolution, said Matthew Baker.

As many as 25 percent of all structures imaged by electron cryo-microscopy and one-third of large protein complexes solved by X-ray crystallography are in the 3 to 10 angstroms range, said Matthew Baker.

Until now, the methodology used to annotate or trace the structure of protein from these density maps was usually tailored to specific cases, said Mariah Baker.

"They involved a lot of user intervention and the possibility to include bias," she said. That sparked a determination to automate the process with better routines that required less specific information.

"The question we asked was, can we trace a protein fold in a density map without a priori knowledge," she said. "The answer is that we can."

Provided by Baylor College of Medicine (news : web)

Protein study gives fresh impetus in fight against superbugs

Researchers have mapped the complex molecular structure of an enzyme found in many bacteria. These molecules – known as restriction enzymes – control the speed at which bacteria can acquire resistance to drugs and eventually become superbugs.

The study, carried out by an international team including scientists from the University of Edinburgh, focused on E. coli, but the results would apply to many other infectious bacteria.

After prolonged treatment with antibiotics, bacteria may evolve to become resistant to many drugs, as is the case with superbugs such as MRSA.

Bacteria become resistant by absorbing DNA – usually from other bugs or viruses – which contains genetic information enabling the bacteria to block the action of drugs. Restriction enzymes can slow or halt this absorption process. Enzymes that work in this way are believed to have evolved as a defence mechanism for bacteria.

The researchers also studied the enzyme in action by reacting it with DNA from another organism. They were able to model the mechanism by which the enzyme disables the foreign DNA, while safeguarding the bacteria's own genetic material. Restriction enzymes' ability to sever genetic material is widely applied by scientists to cut and paste strands of DNA in genetic engineering.

The study was carried out in collaboration with the Universities of Leeds and Portsmouth with partners in Poland and France. It was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust and published in Genes and Development journal.

Dr David Dryden, of the University of Edinburgh's School of Chemistry, who led the study, said: "We have known for some time that these enzymes are very effective in protecting bacteria from attack by other species. Now we have painted a picture of how this occurs, which should prove to be a valuable insight in tackling the spread of antibiotic-resistant superbugs."

Provided by University of Edinburgh

Scientists probe form, function of mysterious protein

Using a combination of laboratory experiments and computer modeling, scientists from Rice University and the University of California, San Diego (UCSD) have deciphered part of mitoNEET's movements to get a better understanding of how it handles its potentially toxic payload of iron and sulfur. Their research is described this week in the Proceedings of the National Academy of Sciences.

"We scrutinize proteins with an unconventional approach," said José Onuchic, Rice's Harry C. and Olga K. Wiess Professor of Physics and Astronomy and co-director of the Center for Theoretical Biological Physics. "We use biophysics to probe biology rather than the other way around. Using computational theory, we find structures that are possible -- regardless of whether they've already been observed experimentally -- and we ask ourselves whether these structures might be biologically significant."

Study co-leader Patricia Jennings, professor of chemistry and biochemistry at UCSD, who has collaborated with Onuchic for 15 years, said they save a great deal of time by using structural biophysics to guide their experiments on a wide variety of targets. For example, Jennings' laboratory determined less than five years ago that mitoNEET contained a novel folded structure. Since then, her lab has been using insights gained from static and dynamic snapshots of the protein to guide biological and biochemical studies.

"I think people forget that proteins are machines with moving parts," said study lead author Elizabeth Baxter, a UCSD graduate student who works under the guidance of both Onuchic and Jennings. "We start with the static snapshot and model in the functional motions."

MitoNEET, which binds to the diabetes drug, Actos, immediately caught the attention of researchers when it was discovered. It has a unique ability to bind and store iron-based molecules in an iron-sulfur cluster. Iron is an essential element for all life, but it is also highly toxic, and mitoNEET is the only iron-handling protein that is known to sit on the wall of the mitochondria, one of the key structures inside a cell.

The protein's biological functions are still being unraveled. Interestingly, scientists have shown that mitoNEET sits on the outer mitochondrial wall with its potentially toxic payload of iron-sulfur molecules facing toward the cell's cytoplasm, the gel-like fluid that fills the cell. Discovery of the unique binding mode of the protein's iron-sulfur cluster led the Jennings group to show that the cluster can be delivered into the mitochondria. In addition, its sister protein interacts with proteins that participate in apoptosis -- the process cells use to kill themselves when they are no longer viable.

"I think mitoNEET is a protein that could be your best friend or your worst enemy," Jennings said. "There's some evidence that it may act as a sensor for oxidative stress and that it can lose its toxic iron-sulfur cluster under stress conditions. Depending upon where the iron ends up, that could lead to drastic problems inside the cell."

Proteins are strands of amino acids that are produced from DNA blueprints, but their shapes can provide important clues about their function. To find out how mitoNEET's control and release of its iron-sulfur payload might be related to its shape, Baxter used computer simulations to study how the protein folds, as well as the functional motions of two similar shapes that could be biologically important. In one of these shapes, there is a slight intertwining of two arms that extend away from the iron-cluster pocket. In the other, the arms also extend but are not intertwined.

Baxter found that both conformations were physically possible. She also found the protein could switch between the "strand-swapped" and "strand-unswapped" conformations without entirely unfolding. Moreover, this change in the twining of the arms was shown to alter the shape of the critical pocket that holds the iron-sulfur cluster; this makes the cluster more likely to be inserted or released in situations where the arms are untwined.

Like the magician using misdirection, the loosening of the grip on the cluster is subtle and happens in a different location than the flurry of arm motions. Jennings said it's the kind of thing that could easily be missed if the focus of the study were the cluster itself.

Onuchic said, "One of the advantages to our approach is that it allows us to look for relevant biophysical properties that control distant functional regions -- like mitoNEET's strand-swapping -- that can easily be missed with a more conventional approach."

More information: http://www.pnas.or … 109.abstract

Provided by Rice University (news : web)

Protein purification alternatives

Protein purification, often referred to as downstream processing, is the most costly and time-consuming process in the manufacture of bio-molecules. EU-funded researchers integrated materials science with process development to produce novel low-cost materials and methods for selective purification with a focus on chromatography, membrane separation and extraction.

Purification is somewhat like passing sand and pebbles through a sieve except that separation is not dependent on gravity and relative size of components and holes. Instead, separation depends on chemical and electrical interactions between the biological fluid and specific binders (ligands) through which it passes.

Among the many proteins purified by the pharmaceutical industry are human immunoglobulin G (IgG) and monoclonal antibodies (MAbs), both important in immunity and thus disease therapy. The most common method for purifying IgG and MAbs is the use of protein A resin. However, pharmaceutical companies are increasingly concerned about the supply of protein A materials.

The 'Advanced interactive materials by design' (AIMS) project thus sought to develop alternatives to protein A technology for the purification of proteins. The investigators developed excellent modelling tools enabling assessment of interactions among support, linker, ligand and product promoting efficient and effective design of new materials.

The researchers created a new SartoAims protein A affinity membrane with enhanced affinity for IgG, providing an important alternative to protein A for IgG purification. In addition, the investigators studied two alternatives to protein A technology for purification of MAbs, one using much less expensive ion exchange resins in a Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) form of chromatography and one using aqueous two-phase extraction.

The researchers also developed new materials for use in ion exchange chromatography, a technique that relies on charge interactions for separation. In fact, the chromatographic resin FractoAims demonstrated superior mechanical stability and can be tailor-made based on bead size, pore size, surface area and ligand density.

The new process concepts were tested in a mini-plant to evaluate performance with respect to protein A technology. A combination of two MCSGP units operating with different parameters enabled reduction in operating costs by a factor of three in total MAb purification costs.

The AIMS project outcomes will have significant impact on the protein purification process that has until now been the most costly part of bio-molecule development in the pharmaceutical, chemical and biotechnology industries. Commercialisation of the new technologies promises to improve the European position in the huge global chemicals and pharmaceuticals market.

Story Source:

The above story is reprinted from materials provided by CORDIS Features, formerly ICT Results, via AlphaGalileo.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

Twenty-year protein mystery solved with surprising results

In spite of more than 20 years of research efforts, the enzymatic function of the CRYM protein has remained elusive. Previous research has shown that CRYM functions both as an important structural protein and a binder of thyroid hormones, but PhD student Andre Hallen suspected something more.

"CRYM was first discovered in the ocular lens of marsupials, that is, in Skippy's eye! Since then, we've seen it in lamb brains, in other tissues and learnt how it can be observed and mutated in mammals like humans. Now we can see more of its full potential in human health and nutrition," Hallen explains.

In a study published in the Journal of Neurochemistry, Hallen conclusively demonstrated an enzyme function for CRYM, and identified how this enzymatic activity reveals a new role for thyroid hormones in regulating mammalian amino acid metabolism.

It also recognises a possible reciprocal role of enzyme activity in regulating bioavailability of intracellular T3, with further research pathways for how this regulatory role might open up new treatment options for a range of neurological and psychiatric conditions.

Hallen lead a team of scientists on this study, including three months working in North America with Dr Arthur Cooper, a world authority on neurochemistry and amino acid chemistry.

His research has also sparked the interest of international scientists, including Patrick W Reed and Robert J Bloch of the University of Maryland, who profiled Hallen's work in their article ‘Crystallin-Gazing: Unveiling Enzymatic Activity'.

In 2012, Hallen will continue his research into this area, further exploring the role of diet in influencing hormone function, and the effects of these changes on the CRYM protein, its related mutations and conditions.

Provided by Macquarie University

Scientists paint new picture of dance between protein and binding partners

Instead, the situation resembles a kind of complex but carefully organized dance routine, where the ligand samples a variety of binding modes while the protein also modifies its shape, a process that results in their pairing and changes in the protein critical for its function.

These new findings, published in the January 11, 2012 edition of the journal Structure, could affect future drug design.

"Using a multidisciplinary approach, we gleaned something from our data that no one else has," said Douglas Kojetin, an assistant professor on the Scripps Florida campus who led the study. "The conventional wisdom is that ligands bind in one orientation but our study shows that they can bind in multiple modes. That means if we can optimize a ligand to bind in mode B rather than mode A, we might be able to select the therapeutic results we want."

The new study—which used a number of complementary technologies including NMR spectroscopy and hydrogen/deuterium exchange (HDX) coupled to mass spectrometery, combined with previous x-ray crystallography analyses—provides detailed insights into the real-time actions of molecules that could never be determined with a single technology.

Specifically, the researchers revealed insights into ligand and receptor dynamics in the nuclear receptor known as PPAR? (peroxisome-proliferator-activated receptor). PPAR? has been implicated in metabolic diseases including obesity, diabetes, and atherosclerosis.

The study also found that various gradations in these ligands influence the dynamics of this exchange, adding another layer of complexity. "One of the compounds, MRL24, binds to the receptor and has anti-diabetic efficacy, but doesn't activate it very well," Kojetin said. "This is what you want because when the receptor is activated you get side effects such as weight gain and brittle bones."

"This study in particular highlights the importance of multidisciplinary collaborative efforts to truly understand the molecular details of drug-receptor interactions", says Kojetin. "This work is an excellent example of the strong campus collaborations we have with the laboratories of Patrick Griffin, Thomas Burris, and Theodore Kamenecka."

More information: The first author of the study, "Ligand and Receptor Dynamics Contribute to the Mechanism of Graded PPAR ? Agonism," is Travis S. Hughes of Scripps Research. Other authors include Michael J. Chalmers, Scott Novick, Dana S. Kuruvilla, Mi Ra Chang, Theodore M. Kamenecka ,Thomas P. Burris, and Patrick R. Griffin of Scripps Research; Mark Rance of the University of Cincinnati; and Bruce A. Johnson of One Moon Scientific Inc.

Provided by The Scripps Research Institute (news : web)

Nanotechnology: Nanomechanical measurements of unprecedented resolution made on protein molecules

UCLA physicists have made nanomechanical measurements of unprecedented resolution on protein molecules.

The new measurements, by UCLA physics professor Giovanni Zocchi and former UCLA physics graduate student Yong Wang, are approximately 100 times higher in resolution than previous mechanical measurements, a nanotechnology feat which reveals an isolated protein molecule, surprisingly, is neither a solid nor a liquid.

"Proteins are the molecular machines of life, the molecules we are made of," Zocchi said. "We have found that sometimes they behave as a solid and sometimes as a liquid.

"Solids have a shape while liquids flow -- for simple materials at low stresses. However, for complex materials, or large stresses, the behavior can be in-between. Subjected to mechanical forces, a material might be elastic and store mechanical energy (simple solid), viscous and dissipate mechanical energy (simple fluid), or visco-elastic and both store and dissipate mechanical energy (complex solid, complex fluid). The viscoelastic behavior characteristic of more complex matter had not been clearly seen before on isolated proteins because mechanical measurements tend to destroy the proteins."

Zocchi and Wang's new nanotechnology method allowed them to apply stresses and probe the mechanics of the protein without destroying it. Wang, now a physics postdoctoral fellow at the University of Illinois in Urbana-Champaign, and Zocchi discovered a "transition to a viscoelastic regime in the mechanical response" of the protein.

"Below the transition, the protein responds elastically, like a spring," Zocchi said. "Above the transition, the protein flows like a viscous liquid. However, the transition is reversible if the stress is removed. Functional conformational changes of enzymes (changes in the shape of the molecule) must typically operate across this transition."

The measurements were performed on the enzyme guanylate kinase, or GK, a member of an essential class of enzymes called kinases. Specifically, GK transfers a phosphate group from ATP (the universal "fuel" of the cell) to GMP, producing GDP, an essential metabolic component, Zocchi said.

The study on the characterization of the "visco-elastic transition" is reported this month in the online journal PLoS ONE, a publication of the Public Library of Science. The research was federally funded by the National Science Foundation's division of materials research and by a grant from the University of California Lab Research Program.

Zocchi and Wang published related findings earlier this year in the journal Europhysics Letters, a publication of the European Physical Society, and the journal Physical Review Letters.

In previous research, Zocchi and colleagues reported a significant step in controlling chemical reactions mechanically last year, made a significant step toward a new approach to protein engineering in 2006, created a mechanism at the nanoscale to externally control the function and action of a protein in 2005, and created a first-of-its-kind nanoscale sensor using a single molecule less than 20 nanometers long in 2003. A nanometer is roughly 2,000 times smaller than the width of a human hair.

The above story is reprinted from materials provided by University of California - Los Angeles. The original article was written by Stuart Wolpert.

Note: Materials may be edited for content and length. For further information, please contact the source cited above.

Journal Reference:

Yong Wang, Giovanni Zocchi. Viscoelastic Transition and Yield Strain of the Folded Protein. PLoS ONE, 2011; 6 (12): e28097 DOI: 10.1371/journal.pone.0028097

Note: If no author is given, the source is cited instead.

Proteins in focus: Adjustable protein microlenses made by femtosecond laser direct writing

Proteins are potentially useful as “building materials” for microcomponents because they are readily available, inexpensive, and biocompatible. They can also change their properties in response to external stimuli, which makes them an interesting material for use in adjustable microlenses. However, lenses must be extremely precise in order to meet optical requirements—something difficult to achieve with proteins. In addition, they must be fast, simple, and inexpensive to produce.

The Chinese researchers have now met this challenge: They used a laser to “write” the desired micrometer-sized lens shape out of a solution of bovine serum albumin, a protein. Methylene blue acts as a photosensitizer, which captures the light energy like an antenna and triggers a crosslinking reaction of the protein molecules. Driven by a computer, the laser cuts out the desired three-dimensional form voxel by voxel. A voxel is a three-dimensional pixel, a tiny segment of volume. The irradiation used is in femtosecond pulses, which lasts on the order of 10-13 seconds. The crosslinking reaction only takes place in the locations that are irradiated. After the reaction, the protein molecules that have not reacted can simply be rinsed away. What stays behind is a cross-linked, aqueous protein gel in the shapes of micrometer-sized lenses.

Direct writing with lasers usually results in structures that have too rough a surface for optical applications. By optimizing the duration of the laser pulse, the pulse intensity, and the protein concentration, Sun and his team obtained lenses with outstanding optical properties.

The special trick in this case is that the amount of liquid absorbed by the protein gel depends on the pH value of the solution. Increasing the pH causes the lens to swell. If the increase in thickness is limited by a glass surface, the lens primarily grows in width and becomes flatter. If the pH value is reduced, the gel shrinks and the lens is more curved. Because the radius of curvature determines the focal length of the lens, this method can be used to focus the microlenses.

Because the protein lenses are biocompatible, they may be used in optical analytical systems for medical diagnostics or lab-on-a-chip technology.

More information: Hong-Bo Sun, Dynamically Tunable Protein Microlenses, Angewandte Chemie International Edition, http://dx.doi.org/ … ie.201105925

Provided by Wiley (news : web)

Soy is on top as a high-quality plant protein

Traditional methods for determining protein quality have shown animal proteins such as milk and eggs to be high in quality. However, those who are interested in a plant-based diet, or diversifying their proteins, have a more difficult time determining which of their choices are high in quality. Testing methods have shown most plant proteins, such as pea protein, are lower in quality than animal-based proteins.

"Accurate methods for determining protein quality are key to helping people plan a healthful diet," said Glenna Hughes, MS, research scientist at Solae. "Due to the increasing interest in including plant-based proteins in the diet, accurate information on protein quality is needed in scientific literature to help educate consumers and healthcare professionals on this topic."

The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) recommend using the protein digestibility-corrected amino acid score (PDCAAS) as a simple and scientific procedure for assessing protein quality. The PDCAAS methodology focuses on three different parameters: the amount of each essential amino acid the protein contains, how easily the protein can be digested, and by taking both of those parameters into account, whether the protein meets the FAO/WHO's amino acid requirements set for children aged two to five years, as they have higher needs to support growth and development than adults.

According to this study, soy protein has a PDCAAS of 1.00, meaning it is a high-quality protein that meets the needs of both children and adults. Eggs, dairy and meat proteins also have a PDCAAS score of 1.0.

However, soy protein is the only widely available high-quality plant-based protein that achieves this score.

"It's important for people to understand that a plant-based diet is healthy, but that not all proteins are created equal," said Connie Diekman, RD, LD, FADA. "If you are planning a vegetarian diet or want to incorporate plant-based proteins in your diet, understanding protein quality using the PDCAAS scale can allow you to select proteins that score higher, such as soy, to ensure that you are getting the essential amino acids you need."

More information: For more information on the study, the following is a link to the abstract: http://www.ncbi.nl … med/22017752

Provided by Solae, LLC

Supercomputer reveals new details behind drug-processing protein model

Jerome Baudry and Yinglong Miao, who are jointly affiliated with ORNL and the University of Tennessee, have performed simulations to observe the motions of water molecules in a class of enzymes called P450s. Certain types of P450 are responsible for processing a large fraction of drugs taken by humans.

The supercomputer simulations were designed to help interpret ongoing neutron experiments.

"We simulated what happens in this enzyme over a time scale of 0.3 microseconds, which sounds very fast, but from a scientific point of view, it's a relatively long time," Baudry said. "A lot of things happen at this scale that had never been seen before. It's a computational tour de force to be able to follow that many water molecules for that long."

The team's study of the water molecules' movements contributes to a broader understanding of drug processing by P450 enzymes. Because some populations have a slightly different version of the enzymes, scientists hypothesize that mutations could partially explain why people respond differently to the same drug. One possibility is that the mutations might shut down the channels that bring water molecules in and out of the enzyme's active site, where the chemical modification of drugs takes place. This could be investigated by using the computational tools developed for this research.

By simulating how water molecules move in and out of the protein's centrally located active site, the team clarified an apparent contradiction between experimental evidence and theory that had previously puzzled researchers. X-ray crystallography, which provides a static snapshot of the protein, had shown only six water molecules present in the active site, whereas experimental observations indicated a higher number of water molecules would be present in the enzyme.

"We found that even though there can be many water molecules -- up to 12 at a given time that get in and out very quickly -- if you look at the average, those water molecules prefer to be at a certain location that corresponds to what you see in the crystal structure," Miao said. "It's a very dynamic hydration process that we are exploring with a combination of neutron scattering experiments and simulation."

The simulation research is published in Biophysical Journal as "Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process."

Provided by Oak Ridge National Laboratory (news : web)

TACC supercomputers help researchers find deeper insight into structure and behavior of protein, DNA and RNA

Analytical ultracentrifugation (AUC) experiments spin samples at very high speeds to study how large molecules such as proteins, DNA and RNA, act in solution. Under the influence of centrifugal forces up to 250,000 times as strong as Earth's gravity, materials undergo sedimentation and diffusion processes over time, revealing aspects of the individual molecules' natures.

These processes are essential measurements for biochemists: a way to understand how molecules behave under physiological solution conditions. And 85 years later, scientists are still finding ways to make the analytic ultracentrifuge more useful.

Unlike traditional microscopy where samples are bound to a microscope grid, or x-ray crystallography where they are locked into a crystal with packing forces that may distort the molecule, AUC experiments preserve the native structures and configurations of molecules. They do this by analyzing molecules in solution, where they can dynamically interact and bind to other molecules, or react to environmental changes such as temperature, ionic strength or pH.

Enlarge

A van Holde - Weischet analysis providing a differential (green) and integral (red) sedimentation profile for a sedimentation velocity experiment performed on a restriction digest of a DNA fragment. In the integral distribution, the vertical axis indicates the relative concentration of each species, while the horizontal axes indicates the sedimentation coefficient.

"If you don't have a way to measure your molecule in solution, then a lot of this will escape you," said Borries Demeler, associate professor of biochemistry at The University of Texas Health Sciences Center and director of the Center for Analytical Ultracentrifugation of Macromolecular Assemblies (CAUMA). "By studying biological macromolecules in a solution, it is possible to observe reactions, and follow conformational changes."

AUC is also a very versatile tool to study composition. Even trace amounts of impurities can be resolved by AUC, and mixtures can be analyzed to identify molecular weight and shape distributions.

Initially, the analysis of centrifugal experiments was done manually, but with the emergence of computers and sensors in the 1960s, more precise ways of assessing experimental results were developed. Today's optical systems can follow sedimenting and diffusing molecules by detecting ultraviolet and visible absorption, the refractive index, and fluorescence emission. The signals are captured digitally to allow them to be analyzed by computer.

For more than two decades, Demeler has worked at the intersection of the physical (spinning samples) and the virtual (supercomputer simulations), investigating new methods and developing software to help researchers make the most of their AUC experiments.

As the director of CAUMA, Demeler works with hundreds of investigators around the world, including biophysicists studying the structure and function of biological macromolecules and assemblies, material scientists trying to make more efficient solar cells, and the pharmaceutical industry evaluating the stability of their formulations. As a collaborator in many research projects, he is continually challenged by new research questions and enjoys the interactions with many fascinating scientists.

His largest impact, however, is felt through the creation of the UltraScan software package, and the development of the UltraScan LIMS portal through which researchers analyze their experimental data over the web using advanced computing methods and systems.

"I started writing the very first version of UltraScan using BASIC on a 286 PC back in 1988," Demeler recalled, "and it's gone through many iterations. "

In 2004, Demeler and his colleague, Emre Brookes, began parallelizing the code so it could run on large-scale computer clusters. This dramatically sped up the rate at which samples could be analyzed. It also enabled the researchers to develop high-resolution analysis methods and address an entirely new class of research questions that widened the application of the AUC method.

Enlarge

A single scan of a sedimentation velocity experiment with semiconducting nanoparticles collected with a novel multiwavelength detector. This novel detector can resolve not only composition by hydrodynamic separation, but also by spectral decomposition, and is developed in Dr. Helmut C?lfen's laboratory at the University of Konstanz in Germany.

UltraScan doesn't just allow researchers to measure the diffusion and sedimentation processes; it decodes the meaning of these processes and uncovers hidden characteristics of the sample.

"We often don't know what really is in a solution provided by a collaborator, and we need to get the most out of our analysis," Demeler explained. "To fit the data, we simulate many different components that may be in the solution, and ask the question, ‘How much of each component is present in the actual experiment?'"

This process can be done on a regular computer, but the answers that such a process generates lack the resolution required for clinical or industrial investigations, or simply take too long to complete.

"To squeeze out the last drop of information, you need to go through quite a bit more computational expense," Demeler said. "This is where we kick in with our methods."

UltraScan's numerical methods extract noise, narrow the parameter space, compare multiple experiments, and determine the uncertainty of the result.

While some analyses are performed on a small development cluster in Demeler's lab, the capacity is insufficient to address the most challenging problems, and to satisfy all of the demand for analysis among a growing international group of AUC users. Instead, Demeler relies on the computing systems of the National Science Foundation funded Extreme Science and Engineering Discovery Environment (XSEDE), the most powerful, and robust collection of integrated advanced digital resources and services in the world.

Demeler's simulations use anywhere between 40 and 14,000 processors simultaneously, speeding up the analytic processing by as much as 10,000 times. In 2010-2011, Demeler used 3.5 million computing hours on the Ranger and Lonestar supercomputers at the Texas Advanced Computing Center (TACC) to perform simulations for the open science community.

"It's not just reserved for biochemists and biophysicists," Demeler said. "We might work with a clinician, perform measurements for materials science, or measure the binding strength of a new drug to its target."

Demeler pointed to a recent example of work he is doing with researchers in Germany characterizing fluorescent nanoparticles made out of cadmium telluride crystals for use in solar panels. Using a new detector developed by a collaborator at the Max Planck Institute, he was able to not only measure the hydrodynamic properties, but also observe their individual absorption spectra, and correlate absorbance properties with particle size.

Enlarge

2-dimensional spectrum - Monte Carlo analysis of a sedimentation velocity experiment. In this experiment two interacting proteins (A and B) were titrated against each other, resulting in a non-globular complex. In the upper left, protein A is shown to generate several globular oligomers. Addition of protein B results in the appearance of a non-globular peak (upper right). An increasing amount of protein B causes an increase of the AB complex (lower left). At the highest concentration of protein B, the lowest molecular weight form of the globular species has completely disappeared, and been converted into the non-globular species. This work was performed in collaboration with Bettie Sue Master's laboratory at UTHSCSA.

Whether the application is nanoparticles for industry or biomarkers in blood, AUC together with UltraScan is an incredibly useful tool. But creating the software and algorithms wasn't the final step. Many potential users of AUC and UltraScan are not computer scientists, and Demeler believed a fear of the supercomputer command line interface would prevent them from using the software.

"To get people to adopt this technology, you have to make it easy for them," he said. "It needs to be extremely robust, user-friendly and intuitive."

Through an Advanced Support for TeraGrid Applications (ASTA) grant from the NSF, staff at Indiana University helped Demeler develop a web-based gateway where researchers log in, access their data, and submit jobs as if they were running a very simple web application.

"The user only has to be familiar with the basic analysis procedure and a web browser; familiarity with Unix supercomputing is not required," Demeler said. "Our users really like this approach."

Eighty-five years after its inception, the evolution of the analytic ultracentrifuge continues. The latest challenge involves finding ways to integrate AUC results with results from other solution methods.

Demeler and Brookes are developing an integrated system combining molecular dynamics with hydrodynamic and small angle scattering simulations to screen a large variety of structural conformations against experimental data. This will give a large pool of researchers new insight into the structure and function of molecules under study.

"The knowledge obtained should enhance our understanding of biomolecular processes, including disease processes, which can lead to improved prevention and treatment," said Emre Brookes. "None of this would be feasible without the vast computational resources available through XSEDE."

Demeler and Brookes' long-term dream is to create a way to integrate all known observational methods — including x-ray crystallography, nuclear magnetic resonance imaging, and calorimetry — to see more deeply than we currently do, without losing sight of the natural conditions under which molecules exist.

"It's like taking a picture of an object from many different angles, and every time you take a picture you see something else that adds to the whole," Demeler said. "By combining them all, the new picture will tell you something you didn't know before."

More information: To read more about the challenges of biochemistry and the role of AUC in protein study, see Demeler's recent commentary in the June 2011 edition of Nature Chemical Biology.

The story is courtesy of Faith Singer-Villalobos @ Texas Advanced Computing Center (TACC).

Provided by University of Texas at Austin (news : web)

New protein structure expands nature's repertoire of biomolecules

The artificial protein made by the Bristol team – which they have named CC-Hex – has 6 polypeptide chains that the team designed from first principles; that is, whilst the chains take inspiration from biology they are not based on or related to any one particular natural protein.  Each chain folds into a helix, and these assemble to form a bundle (see top image).

This is interesting because nature appears not to have used this structure, or at least natural analogues of CC-Hex have not yet been observed.  Moreover, the structure is intriguing as the helices come together to form a ring that defines a central channel (see middle and bottom images).

 The protein has a central channel with defined chemistry that can be altered and controlled

This central channel provides the basis for engineering new proteins such as ion channels, which may be used as components of sensor and purification devices, and catalysts, which could pave the way to new industrial enzymes

The team, led by Professor Dek Woolfson and Professor Leo Brady, has also shown that the chemistry inside the channel can be altered using further design, chemical synthesis and X-ray crystallography.

Despite quite radical changes to the internal chemistry, the protein structure is robust to these alterations.  This is exciting because it is precisely how many natural proteins function: they alter chemistry within defined and highly controlled cavities within protein structures.  With this in mind, the team believes that CC-Hex represents an exciting opportunity to design new proteins, including enzymes and ion channels, from scratch.

Professor Dek Woolfson said of the discovery: “This is an exciting time for our labs.  Not only have we found a part of protein space that nature seems to have neglected, but we believe that the new structure will allow us to engineer protein functions much more rationally and confidently than has been possible before.”

More information: ‘A de novo peptide hexamer with a mutable channel’ by NR Zaccai, B Chi, AR Thomson, AL Boyle, GJ Bartlett, M Bruning, N Linden, RB Sessions, PJ Booth, RL Brady, and DN Woolfson in Nat. Chem. Biol. DOI: 10.1038/NChemBio.692

Provided by University of Bristol (news : web)

First detection of pregnancy protein in older people destined for Alzheimer's disease

 

In an advance toward a much-needed early diagnostic test for Alzheimer's disease (AD), scientists have discovered that older women destined to develop AD have high blood levels of a protein linked to pregnancy years before showing symptoms. Their report appears in ACS' Journal of Proteome Research.

Theo Luider and colleagues explain that more than 26 million people worldwide already have AD, and the numbers are rising with the graying of the population. Doctors can prescribe any of several drugs to slow the disease's advance. But it is important to start treatment as early as possible. Unfortunately, however, no test exists to diagnose patients before obvious memory loss and other symptoms appear. Luider's team decided to look for proteins in the blood that might be used in such a test.

They looked for those proteins in blood samples of 86 people aged 60-90 who participated in a larger study of aged-related brain changes conducted in The Netherlands. Surprisingly, Luider's group found that significant elevations in pregnancy zone protein (PZP) occurred in women an average of 4 years before diagnosis of AD. Scientists long have known that PZP levels rise during pregnancy, but this was the first link with AD. Luider further discovered the apparent source of the PZP in the brain of these women, who were not pregnant: PZP was being produced in senile plaques, degenerated areas of the brain associated with AD.

More information: “Serum Levels of Pregnancy Zone Protein Are Elevated in Presymptomatic Alzheimer’s Disease” J. Proteome Res., Article ASAP. DOI: 10.1021/pr200270z

Abstract
We have sought for disease-related proteins that could predict the onset of Alzheimer’s disease (AD) in a study population derived from the Rotterdam Scan Study, a population-based prospective cohort study designed to investigate the etiology and natural history of age-related brain changes in the elderly. The serum proteome of 43 persons who developed AD, after an average of 4.2 years (±2.6 years SD) after blood sampling, and 43 gender- and age-matched controls who remained dementia-free during follow-up was investigated by liquid chromatography mass spectrometry. We identified 61 differentially expressed peptides between presymptomatic AD and controls, 9 of which were derived from pregnancy zone protein (PZP). Quantitative measurements using a multiple reaction monitoring assay showed a significant increase in concentration of PZP in presymptomatic AD (34.3 ± 20.6 mg/L) compared with controls (23.6 ± 13.6 mg/L) (p = 0.006). The difference in PZP was significant in women. Immunohistochemical validation of the findings on brain tissue sections showed strong PZP expression in senile plaques and in microglial and glial cells in AD with only low expression in some scattered glial cells in controls.

Provided by American Chemical Society (news : web)

Compound kills highly contagious flu strain by activating antiviral protein

A compound tested by UT Southwestern Medical Center investigators destroys several viruses, including the deadly Spanish flu that killed an estimated 30 million people in the worldwide pandemic of 1918.

This lead compound - which acts by increasing the levels of a human antiviral protein - could potentially be developed into a new drug to combat the flu, a virus that tends to mutate into strains resistant to anti-influenza drugs.

"The virus is 'smart' enough to bypass inhibitors or vaccines sometimes. Therefore, there is a need for alternative strategies. Current drugs act on the virus, but here we are uplifting a host/human antiviral response at the cellular level," said Dr. Beatriz Fontoura, associate professor of cell biology and senior author of the study available online in Nature Chemical Biology.

According to National Institutes of Health, influenza hospitalizes more than 200,000 people in the U.S. each year, with about 36,000 fatalities related to the illness. Worldwide, flu kills about 500,000 people annually.

In the latest cell testing, the compound successfully knocked out three types of influenza as well as a smallpox-related virus and an animal virus. Because of the highly contagious nature of the 1918 flu, those tests took place at Mount Sinai School of Medicine in New York, one of the few places that stores and runs tests on that flu strain.

The compound is among others that the research team is testing that induce an infection-fighting human protein called REDD1. Until this study, researchers had not demonstrated that REDD1 had this important antiviral function.

"We've discovered that REDD1 is a key human barrier for infection," said Dr. Fontoura, "Interestingly, REDD1 inhibits a signaling pathway that regulates cell proliferation and cancer."

The UT Southwestern-led research team tested 200,000 compounds for those that would inhibit flu virus infection. A total of 71 were identified.

Using the two most promising compounds, researchers at UT Southwestern and colleagues at Mount Sinai next will work to strengthen their potencies for further testing. Dr. Fontoura said it can take more than 10 years before successful compounds are developed into drugs.

Provided by UT Southwestern Medical Center (news : web)

From protein to planes and pigskin

Scientists may soon be able to make pest insects buzz off for good or even turn them into models for new technologies, all thanks to a tiny finding with enormous potential.

Sujata Chaudhari, a Kansas State University doctoral candidate in biochemistry, Pune, India, is the senior author of a study that was published this week in the Proceedings of the National Academy of Sciences. Her work includes a discovery that could expand the possibilities for selective pest control and new biomaterials like football padding or lightweight aircraft components -- and all by debunking a more than 50-year-old belief about the protective shell of insects.

The study looks at the red flour beetle and examines the dynamic biochemical processes the insect uses to replace the protective coating on its skin while shedding its old skin. This coating is called the cuticle and is the main structural and protective part of an insect's exoskeleton, creating a stiff but lightweight outer shell or flexible wings and joints.

"As an insect develops, it outgrows its rigid skin and must periodically get rid of its old cuticle and synthesize a new, larger one," Chaudhari said. "This process of shedding the old cuticle is called molting."

In order to molt, the insect's body secretes a fluid loaded with an enzyme called chitinase, which is pronounced ky-tin-ayes. Chitinase breaks down chitin, the main component of the cuticle, and consequently aids in dissolving the insect's old cuticle. For decades it has been assumed that chitinase does not come into contact with and dissolve the insect's newly formed cuticle because of an impenetrable envelope between the old and new cuticles, Chaudhari said.

But Chaudhari and her colleagues found that's not actually the case.

Instead, their research shows that chitinase is present in the new cuticle as well as in the old cuticle. Moreover, they found that the enveloping layer that separates the two cuticles is not responsible for protecting the new cuticle from being dissolved by chitinase. Rather it is the protein called Knickkopf -- pronounced kuh-NICK-kaw-pff.

"Think of Knickkopf as a fire retardant, chitinase as a fire, and the insect's cuticle as the wall of a house," said Subbaratnam Muthukrishnan, a university distinguished professor of biochemistry at Kansas State University, Chaudhari's adviser and a collaborator on the study. "During molting, it's like the house is on fire, but the fire is only burning things on the outside. Everything inside is safe because there's a fire retardant wall."

Although this discovery that chitinase is stopped by a protein and not a physical barrier was made in the red flour beetle, Tribolium castaneum, the same protein is found in all other insect species examined, and probably has the same chitin-protective function, Chaudhari said. Most likely the same holds true for all arthropods: insects, arachnids, crustaceans, nematodes and other organisms. That's a game-changer for scientists and inventors.

In the future, agricultural crop pests like the red flour beetle could find themselves the targets of insecticides or interfering RNAs that shut down the Knickkopf protein, leaving the insect's body open to disease or to molting defects, said Richard Beeman, a Kansas State University entomology adjunct professor, researcher with the U.S. Department of Agriculture and collaborator on the project. Additionally, the beetle's cuticle could be replicated into new lightweight body armor, prosthetics or materials for flight.

"The cuticle is a gigantic puzzle, and we're slowly finding what the pieces are in the puzzle and how they interact to make the cuticle, organize it and digest it," said Karl Kramer, a Kansas State University emeritus biochemistry adjunct professor and collaborator with the USDA, who also worked on the project. "In solving the puzzle, we could target these composition materials for improved insect control. We could also develop biomaterial that could be used in agriculture or medicine -- or even make K-State football coach Bill Snyder some new protective padding for the Wildcats."

More information: "Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton," Proceedings of the National Academy of Sciences.

Provided by Kansas State University (news : web)

Scientists pinpoint shape-shifting mechanism critical to protein signaling

In a joint study, scientists from the California and Florida campuses of The Scripps Research Institute have shown that changes in a protein's structure can change its signaling function and they have pinpointed the precise regions where those changes take place.

The new findings could help provide a much clearer picture of potential drugs that would be both effective and highly specific in their biological actions.

The study, led by Patrick Griffin of Scripps Florida and Raymond Stevens of Scripps California, was published in a recent edition of the journal Structure.

The new study focuses on the ß2-adrenergic receptor, a member of the G protein-coupled receptor family. G protein-coupled receptors convert extracellular stimuli into intracellular signals through various pathways. Approximately one third of currently marketed drugs (including for diabetes and heart disease) target these receptors.

Scientists have known that when specific regions of the receptor are activated by neurotransmitters or hormones, the structural arrangement (conformation) of the receptor is changed along with its function.

"While it's accepted that these receptors adopt multiple conformations and that each conformation triggers a specific type of signaling, the molecular mechanism behind that flexibility has been something of a black box," said Griffin, who is chair of the Scripps Research Department of Molecular Therapeutics and director of the Scripps Florida Translational Research Institute. "Our findings shed significant light to it."

The study describes in structural detail the various regions of the receptor that are involved in the changes brought about by selective ligands (ligands are molecules that bind to proteins to form an active complex), which, like a rheostat, run the gamut among activating the receptor, shutting it down, and reversing its function, as well as producing various states in between.

To achieve the results described in the study, the team used hydrogen-deuterium (HDX) mass spectrometry to measure the impact of interaction of various functionally selective ligands with the ß2-adrenergic receptor. A mass spectrometer determines the mass of fragments from the receptor by measuring the mass-to-charge ratio of their ions. HDX has been used to examine changes in the shape of proteins and how these shape changes relate to protein function. The approach is often used to characterize protein-protein interactions that are critical for signal transduction in cells and to study protein-folding pathways that are critical to cell survival.

"At this early stage in understanding GPCR structure and function, it is important to view the entire receptor in combination with probing very specific regions," said Stevens, who is a professor in the Scripps Research Department of Molecular Biology. "Hydrogen-deuterium exchange mass spectrometry has the right timescale and resolution to asked important questions about complete receptor conformations in regards to different pharmacological ligand binding. The HDX data combined with the structural data emerging will really help everyone more fully understand how these receptors work."

"Using the HDX technology we can study the intact receptor upon interaction with ligands and pinpoint regions of the receptor that have undergone change in position or flexibility," Griffin said. "By studying a set of ligands one can start to develop patterns that are tied to activation of the receptor or shutting it down. Once we get a picture of what a functional ligand looks like, it might be possible to develop a drug to produce a highly selective therapeutic effect."

More information: "Ligand-Dependent Perturbation of the Conformational Ensemble for the GPCR b2 Adrenergic Receptor Revealed by HDX," Structure.

Provided by The Scripps Research Institute (news : web)