From U Alberta: “Allergic drug reactions traced to single protein”

University of Alberta

February 2, 2015
Ross Neitz

Research from UAlberta and Johns Hopkins University points to new strategy to reduce allergic responses to multiple medications.

Every day in hospitals around the world, patients suffer painful allergic reactions to the medicines they are given. The reactions, known as pseudo-allergies, often cause patients to endure itchiness, swelling and rashes as an unwanted part of their treatment plan. The reactions can be so severe they may stop patients from taking their needed medications and sometimes can even prove fatal. It’s never been shown conclusively what triggers these allergic reactions—until now.

“We are in the very early stages but we now understand how these pseudo-allergies are happening,” says Marianna Kulka, an adjunct assistant professor in the University of Alberta’s Department of Medical Microbiology and Immunology and project group leader with the National Institute for Nanotechnology. “This is a very large step forward in many ways.”

In a study published in the December edition of the journal Nature, researchers from the U of A’s Faculty of Medicine & Dentistry and Johns Hopkins University identified a single protein as the root cause of allergic reactions to drugs and injections. They are now exploring ways to block the protein and reduce painful side effects caused by the reactions.

“The drugs currently being used are to treat some very nasty diseases and they’re very effective at that. But side effects are a huge problem. If we can avoid these side effects by finding a way to block this problematic protein, we can really design drugs that are effective and safe,” says Kulka, a co-author on the study.

In their findings the researchers focused on reactions triggered by medicines prescribed for a number of conditions that range from prostate cancer to diabetes to HIV. These reactions are different from the allergic reactions caused by food or experienced by hay fever sufferers.

The scientists tested lab models with and without a single protein—named MRGPRB2—on their cells. The lab models without the protein did not suffer negative effects despite being given drugs known to provoke reactions.

Benjamin McNeil, a post-doctoral fellow at Johns Hopkins University and study co-author, says, “It’s fortunate that all of the drugs turn out to trigger a single receptor—it makes that receptor an attractive drug target.”

The researchers say if a new drug to block the protein receptor could be made, it would lessen the drug side-effects patients currently endure. Kulka believes with time, some painful reactions from medications can be avoided.

“By understanding how they’re happening we can really help to avoid some of the pitfalls of designing drugs that cause the pseudo-allergies. We’ve got big plans in the future for trying to expand this [research] and better understand how this works.”

Research funding was provided by the Canadian Institutes of Health Research and the National Institutes of Health.

Other authors on the paper are Priyanka Pundir, a post-doctoral fellow with the U of A, and Sonya Meeker, Liang Han, Bradley J. Undem and Xinzhong Dong of Johns Hopkins University.

See the full article here.

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UAlberta’s daring and innovative spirit inspires faculty and students to advance knowledge through research, seek innovation in teaching and learning, and find new ways to serve the people of Alberta, the nation, and the world.

The University of Alberta’s has had the vision to be one of the world’s great universities for the public good since its inception. This university is dedicated to the promise made by founding president Henry Marshall Tory that “… knowledge shall not be the concern of scholars alone. The uplifting of the whole people shall be its final goal.”

From UC Irvine: “Chemists find a way to unboil eggs”

UC Irvine

January 23, 2015
Janet Wilson, UC Irvine

UC Irvine and Australian chemists have figured out how to unboil egg whites — an innovation that could dramatically reduce costs for cancer treatments, food production and other segments of the $160 billion global biotechnology industry, according to findings published today in the journal ChemBioChem.

“Yes, we have invented a way to unboil a hen egg,” said Gregory Weiss, UCI professor of chemistry and molecular biology & biochemistry. “In our paper, we describe a device for pulling apart tangled proteins and allowing them to refold. We start with egg whites boiled for 20 minutes at 90 degrees Celsius and return a key protein in the egg to working order.”

Like many researchers, he has struggled to efficiently produce or recycle valuable molecular proteins that have a wide range of applications but which frequently “misfold” into structurally incorrect shapes when they are formed, rendering them useless.

“It’s not so much that we’re interested in processing the eggs; that’s just demonstrating how powerful this process is,” Weiss said. “The real problem is there are lots of cases of gummy proteins that you spend way too much time scraping off your test tubes, and you want some means of recovering that material.”

But older methods are expensive and time-consuming: The equivalent of dialysis at the molecular level must be done for about four days. “The new process takes minutes,” Weiss noted. “It speeds things up by a factor of thousands.”

To re-create a clear protein known as lysozyme once an egg has been boiled, he and his colleagues add a urea substance that chews away at the whites, liquefying the solid material. That’s half the process; at the molecular level, protein bits are still balled up into unusable masses. The scientists then employ a vortex fluid device, a high-powered machine designed by professor Colin Raston’s laboratory at South Australia’s Flinders University. Shear stress within thin, microfluidic films is applied to those tiny pieces, forcing them back into untangled, proper form.

“This method … could transform industrial and research production of proteins,” the researchers write in ChemBioChem.

For example, pharmaceutical companies currently create cancer antibodies in expensive hamster ovary cells that do not often misfold proteins. The ability to quickly and cheaply re-form common proteins from yeast or E. coli bacteria could potentially streamline protein manufacturing and make cancer treatments more affordable. Industrial cheese makers, farmers and others who use recombinant proteins could also achieve more bang for their buck.

UCI has filed for a patent on the work, and its Office of Technology Alliances is working with interested commercial partners.

Besides Weiss and Raston, the paper’s authors are Tom Yuan, Joshua Smith, Stephan Kudlacek, Mariam Iftikhar, Tivoli Olsen, William Brown, Kaitlin Pugliese and Sameeran Kunche of UCI, as well as Callum Ormonde of the University of Western Australia. The research was supported by the National Institute of General Medical Sciences and the Australian Research Council.

See the full article here.

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Since 1965, the University of California, Irvine has combined the strengths of a major research university with the bounty of an incomparable Southern California location. UCI’s unyielding commitment to rigorous academics, cutting-edge research, and leadership and character development makes the campus a driving force for innovation and discovery that serves our local, national and global communities in many ways.

With more than 29,000 undergraduate and graduate students, 1,100 faculty and 9,400 staff, UCI is among the most dynamic campuses in the University of California system. Increasingly a first-choice campus for students, UCI ranks among the top 10 U.S. universities in the number of undergraduate applications and continues to admit freshmen with highly competitive academic profiles.

UCI fosters the rigorous expansion and creation of knowledge through quality education. Graduates are equipped with the tools of analysis, expression and cultural understanding necessary for leadership in today’s world.

Consistently ranked among the nation’s best universities – public and private – UCI excels in a broad range of fields, garnering national recognition for many schools, departments and programs. Times Higher Education ranked UCI No. 1 among universities in the U.S. under 50 years old. Three UCI researchers have won Nobel Prizes – two in chemistry and one in physics.

The university is noted for its top-rated research and graduate programs, extensive commitment to undergraduate education, and growing number of professional schools and programs of academic and social significance. Recent additions include highly successful programs in public health, pharmaceutical sciences and nursing science; an expanding education school; and a law school already ranked among the nation’s top 10 for its scholarly impact.

From UCSC: “Researchers find a novel signaling pathway involved in appetite control”

UC Santa Cruz

January 19, 2015
Tim Stephens

Agouti-related protein regulates feeding behavior, illustrated here in the Eastern chipmunk. (Photo by Ed Reschke)

A new study has revealed important details of a molecular signaling system in the brain that is involved in the control of body weight and metabolism. The study, published January 19 in Nature, provides a new understanding of the melanocortin pathway and could lead to new treatments for obesity.

Coauthor Glenn Millhauser, a distinguished professor of chemistry and biochemistry at UC Santa Cruz, said the findings are very exciting and have broad biomedical implications. “We are getting to the real molecular features of what’s controlling this important signaling system in the brain,” Millhauser said.

The study, led by researchers at Vanderbilt University, focused on a receptor embedded in the membranes of nerve cells called the melanocortin-4 receptor, or MC4R. It belongs to a class of receptors known as G-protein coupled receptors (GPCRs), which typically act like on-off switches, signaling over short time frames, according to Roger Cone, who led the study at Vanderbilt.

“This finding identifies a molecular mechanism for converting an on-off switch into a rheostat,” Cone said. “This could help explain slow, sustained biological processes that also are mediated by GPCRs, such as tanning or weight regain after dieting.”

Millhauser’s lab has done extensive research on proteins that bind to the MC4R receptor, such as agouti-related protein (AgRP). AgRP is a potent appetite stimulant. Its role in regulating energy balance is to suppress metabolism and increase feeding when the body needs to put on weight and store energy, Millhauser said. His lab has developed modified versions of the AgRP protein that alter its activity. In the new study, the modified proteins from Millhauser’s lab helped researchers identify a previously unsuspected effect of AgRP.

Millhauser’s previous studies have shown that a single dose of AgRP given to laboratory animals can stimulate daily food intake for up to 10 days. This observation didn’t fit with the traditional “on-off” signaling model for the receptor it binds to, MC4R. G-protein coupled receptors can only take so much stimulation before they shut down, and this phenomenon, called desensitization, often happens rapidly.

Cone’s lab discovered a companion protein–a potassium channel in the membrane called Kir7.1–that couples to the MC4R receptor and acts independently from G-protein signaling. The researchers found that AgRP induces MC4R to open the potassium channel, which “hyperpolarizes” and inhibits neurons that are involved in blocking appetite.

“Moreover, with modifications to AgRP discovered previously by our lab, we can increase or decrease this coupling of the receptor to the potassium channel,” Millhauser said. “These concepts could ultimately lead to new generations of therapeutics for treating metabolic disorders, including obesity, anorexia, and cachexia, the wasting condition that often occurs in cancer treatment.”

Coauthor Rafael Palomino, a graduate student and NIH Fellow in Millhauser’s lab, did the protein synthesis and purification work for the study. The first author is Masoud Ghamari-Langroudi at Vanderbilt. Other contributors include Jerod Denton and Robert Matthews at Vanderbilt and Helen Cox at King’s College, London. This research was supported by the National Institutes of Health.

See the full article here.

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The University of California, Santa Cruz, opened in 1965 and grew, one college at a time, to its current (2008-09) enrollment of more than 16,000 students. Undergraduates pursue more than 60 majors supervised by divisional deans of humanities, physical & biological sciences, social sciences, and arts. Graduate students work toward graduate certificates, master’s degrees, or doctoral degrees in more than 30 academic fields under the supervision of the divisional and graduate deans. The dean of the Jack Baskin School of Engineering oversees the campus’s undergraduate and graduate engineering programs.

From SLAC: “X-ray Laser Reveals How Bacterial Protein Morphs in Response to Light”

SLAC Lab

December 4, 2014

A Series of Super-Sharp Snapshots Demonstrates a New Tool for Tracking Life’s Chemistry

Human biology is a massive collection of chemical reactions, from the intricate signaling network that powers our brain activity to the body’s immune response to viruses and the way our eyes adjust to sunlight. All involve proteins, known as the molecules of life; and scientists have been steadily moving toward their ultimate goal of following these life-essential reactions step by step in real time, at the scale of atoms and electrons.

Now, researchers have captured the highest-resolution snapshots ever taken with an X-ray laser that show changes in a protein’s structure over time, revealing how a key protein in a photosynthetic bacterium changes shape when hit by light. They achieved a resolution of 1.6 angstroms, equivalent to the radius of a single tin atom.

This illustration depicts an experiment at SLAC that revealed how a protein from photosynthetic bacteria changes shape in response to light. Samples of the crystallized protein (right), called photoactive yellow protein or PYP, were jetted into the path of SLAC’s LCLS X-ray laser beam (fiery beam from bottom left). The crystallized proteins had been exposed to blue light (coming from left) to trigger shape changes. Diffraction patterns created when the X-ray laser hit the crystals allowed scientists to recreate the 3-D structure of the protein (center) and determine how light exposure changes its shape. (SLAC National Accelerator Laboratory)

“These results establish that we can use this same method with all kinds of biological molecules, including medically and pharmaceutically important proteins,” said Marius Schmidt, a biophysicist at the University of Wisconsin-Milwaukee who led the experiment at the Department of Energy’s SLAC National Accelerator Laboratory. There is particular interest in exploring the fastest steps of chemical reactions driven by enzymes — proteins that act as the body’s natural catalysts, he said: “We are on the verge of opening up a whole new unexplored territory in biology, where we can study small but important reactions at ultrafast timescales.”

The results, detailed in a report published online Dec. 4 in Science, have exciting implications for research on some of the most pressing challenges in life sciences, which include understanding biology at its smallest scale and making movies that show biological molecules in motion.

A New Way to Study Shape-shifting Proteins

The experiment took place at SLAC’s Linac Coherent Light Source (LCLS), a DOE Office of Science User Facility. LCLS’s X-ray laser pulses, which are about a billion times brighter than X-rays from synchrotrons, allowed researchers to see atomic-scale details of how the bacterial protein changes within millionths of a second after it’s exposed to light.

LCLS at SLAC

“This experiment marks the first time LCLS has been used to directly observe a protein’s structural change as it happens. It opens the door to reaching even faster time scales,” said Sébastien Boutet, a SLAC staff scientist who oversees the experimental station used in the study. LCLS’s pulses, measured in quadrillionths of a second, work like a super-speed camera to record ultrafast changes, and snapshots taken at different points in time can be compiled into detailed movies.

The protein the researchers studied, found in purple bacteria and known as PYP for “photoactive yellow protein,” functions much like a bacterial eye in sensing blue light. The mechanism is very similar to that of other receptors in biology, including receptors in the human eye. “Though the chemicals are different, it’s the same kind of reaction,” said Schmidt, who has studied PYP since 2001. Proving the technique works with a well-studied protein like PYP sets the stage to study more complex and biologically important molecules at LCLS, he said.

Chemistry on the Fly

In the LCLS experiment, researchers prepared crystallized samples of the protein, and exposed the crystals, each about 2 millionths of a meter long, to blue laser light before jetting them into the LCLS X-ray beam.

The X-rays produced patterns as they struck the crystals, which were used to reconstruct the 3-D structures of the proteins. Researchers compared the structures of the proteins that had been exposed to light to those that had not to identify light-induced structural changes.

“In the future we plan to study all sorts of enzymes and other proteins using this same technique,” Schmidt said. “This study shows that the molecular details of life’s chemistry can be followed using X-ray laser crystallography, which puts some of biology’s most sought-after goals within reach.”

Researchers from the University of Wisconsin-Milwaukee and SLAC were joined by researchers from Arizona State University; Lawrence Livermore National Laboratory; University of Hamburg and DESY in Hamburg, Germany; State University of New York, Buffalo; University of Chicago; and Imperial College in London. The work was supported by the National Science Foundation, National Institutes of Health and Lawrence Livermore National Laboratory.

See the full article here.

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SLAC is a multi-program laboratory exploring frontier questions in photon science, astrophysics, particle physics and accelerator research. Located in Menlo Park, California, SLAC is operated by Stanford University for the DOE’s Office of Science.

From LBL: “A Cage Made of Proteins, Designed With Help From the Advanced Light Source”

Berkeley Lab

November 19, 2014
Dan Krotz 510-486-4019

With help from Berkeley Lab’s Advanced Light Source, scientists from UCLA recently designed a cage made of proteins.

The nano-sized cage could lead to new biomaterials and new ways to deliver drugs inside cells. It boasts a record breaking 225-angstrom outside diameter, the largest to date for a designed protein assembly. It also has a 130-angstrom-diameter central cavity, which is large enough to hold molecular cargo. And its high porosity is perfect for packing a lot of chemistry in a small package.

More research is needed, but perhaps scientists could some day insert a cancer-fighting drug inside the cage, and tweak its exterior proteins so that it targets malignant cells.

That’s one promise of the new cage. Another is the way in which it was designed. The cage is composed of specially designed “building block” proteins. When the proteins are in a solution with just the right conditions, they assemble into a hollow cube made of 24 proteins. Some of these cubes form crystals.

The scientists used the Advanced Light Source, a synchrotron located at Berkeley Lab, to quickly visualize the cage in different solutions. This helped the scientists determine how to best get the cage to assemble itself. It also allowed them to see how different solutions yield cages of various geometries.

LBL ALS

They used beamline 12.3.1, also known as SIBYLS, which stands for Structurally Integrated Biology for Life Sciences. The SIBYLS beamline is optimized for the joint application of crystallography and SAXS imaging, or small-angle X-ray scattering. SAXS provides information on the shapes of large molecular assemblies in almost any type of solution. And it’s much faster than conventional protein crystallography techniques.

“SAXS helped us efficiently and quickly understand the assembly processes of these protein cages. We had feedback in a matter of hours, not days” says Greg Hura, a scientist with Berkeley Lab’s Physical Biosciences Division.

Hura and John Tainer of Berkeley Lab’s Life Sciences Division are co-authors of a Nature Chemistry paper that describes the protein cage. The research was led by Todd Yeates, a UCLA professor of chemistry and biochemistry.

Greg Hura at the The SYBILS beamline at the Advanced Light Source, which can quickly visualize a protein assembly’s structure in almost any solution, is helping researchers design new biomaterials.

SAXS made its mark elucidating the structure of proteins critical to human health, such as DNA repair machines. The technique can analyze about 100 samples in four hours. It also analyzes samples in solutions that approximate the biological conditions in which proteins are found. Hura and Tainer are now expanding SAXS’s repertoire to assist in the development of biomaterials.

“The magic of proteins is they are capable of a tremendous amount of chemistry, which we can harness in advanced materials for medicine, energy, and other applications,” says Hura, who helped optimize SAXS for high-throughput use.

The technique could be especially useful in helping to integrate the nanoscale properties of individual proteins into large complexes that perform useful functions. For example, Hura envisions using SAXS to develop protein assemblies that act as highly efficient catalysts, complete with millions of points that interact with a substance of choice.

“We are keenly interested in the rules for assembly at these nanoscales, since many alternative and valuable designs are currently being explored,” says Hura.

For the UCLA-developed protein cage, SAXS helped the scientists develop an annealing process that yielded crystal structures of the cage in eight hours. Before, it took several months for crystals to form. SAXS also enabled the team to analyze the protein cages under real-world physiological conditions, such as the pH levels found inside cells, and see how these conditions affected the cages’ properties.

“The technique allows the direct visualization of a structure’s flexibility and variability in solution, which will help improve the design of protein cages and other biomaterials,” says Hura.

See the full article here.

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From APS at Agonne Lab: “Advanced Photon Source to remain leader in protein structure research for years”

News from APS at Argonne National Laboratory

May 5, 2014
Brian Grabowski

Proteins are involved in virtually every process in all living cells on the planet, be it a bacterium or yourself. In humans, antibodies defend against invading bacteria, viruses and other infectious agents. Insulin helps regulate how your body uses carbohydrates and fats. Lactase helps digest lactose from dairy products.

The world’s first protein characterization research facility directly attached to a light source will open in the near future at the Advanced Photon Source. The Advanced Protein Characterization Facility will use state-of-the-art robotics for gene cloning, protein expression, protein purification and protein crystallization.

But scientists know the structures and functions of only a small fraction of the proteins in living systems. The vast majority remain a mystery. The backlog of uncharacterized proteins grows quickly every day as scientists continue to determine the genetic makeups of thousands of new organisms, using astonishingly efficient techniques of genome sequencing.

No X-ray facility in the world has supported more protein structure research and characterized more proteins than the Advanced Photon Source (APS) at the U.S. Department of Energy’s Argonne National Laboratory. Soon this 2/3-mile-in-circumference X-ray instrument will get a boost in efficiency that likely will translate into a big boon for the discovery of new pharmaceuticals and the control of genetic disorders and other diseases, as well as advancing the biotech industry.

The world’s first protein characterization research facility directly attached to a light source will open in the near future at the APS. The Advanced Protein Characterization Facility (APCF) will use state-of-the-art robotics for gene cloning, protein expression, protein purification and protein crystallization.

Not beautiful, but very efficient

“The net result will be more protein structures analyzed per year, higher resolution structures and more research into protein function,” said Andrzej Joachimiak, an Argonne Distinguished Fellow who also is director of the Structural Biology Center’s (SBC’s) Sector 19 beamlines and the Midwest Center for Structural Genomics. “This facility has been designed to integrate systems biology and molecular biology with gene cloning, protein expression, protein purification, protein crystallization and crystal testing and delivery to the APS. There is nothing like this anywhere in the world right now.”

When a new protein structure is discovered and verified, the data are deposited in the Protein Data Bank repository to make it available to researchers around the world. For the last 11 consecutive years, the APS has been far and away the world leader in protein structure deposits. The APS has 14 beamlines dedicated to the study of protein crystals through a technique called macromolecular crystallography.

“Two Nobel Prizes for Chemistry were awarded in the past four years for APS-based research involving crystallography,” said Joachimiak, “One Nobel Prize was for research into the structure and function of ribosomes on SBC’s 19-ID beamline, and another was awarded in 2012 for studies of G-protein-coupled receptors at a GM/CA-CAT micro-focus beamline.”

Ribosomes make proteins in all living cells. Improved knowledge about bacterial ribosomes, for example, is speeding development of new antibiotics that combat bacterial infections by interfering with protein production. G-protein-coupled receptor (GPCR) proteins help cells stay in constant communication with each other, thereby facilitating resource sharing. When normal cells become cancerous, GPCRs are changed, too. The change can corrupt the lines of communication, allowing the cancerous cells to grow without limits. The first discovery of the structure of a human GPCR was made at the APS as part of the Nobel Prize-winning research. In fact, the structure was captured at the exact moment the GPCR was signaling across a cell membrane.

The APCF will be available for use by the more than 5,500 scientists who visit the APS annually, but it will have a particularly strong connection to Argonne’s SBC and the beamline it operates at Sector 19. In 2013, more than 660 crystallographers used the SBC facility to collect data on hundreds of projects, including proteins from the Ebola virus. More than 4,100 protein structures have been deposited into the Protein Data Bank from SBC.

Protein structures are analyzed by crystalling the proteins and then placing the single crystals into an X-ray beam for analysis using X-ray diffraction. The results depend on the quality of both the protein crystal and the X-ray beam. The APS provides some of the most brilliant X-ray beams in the Western Hemisphere. Additionally, the APS generates a highly parallel beam, which enables tight focusing of the X-rays. Staff at the National Institute of General Medical Sciences and National Cancer Institute structural biology facility (GM/CA-CAT) beamline capitalized on this and created the world’s first micro X-ray beam at the request of visiting researchers. “The micro-beam was essential for the GPCR research,” said Joachimiak.

The crystallography capabilities of the APS will increase with a planned upgrade. “After the upgrade, the brilliance of the X-ray beam will increase by two to three orders of magnitude,” Joachimiak said. “The beam will be more parallel, too, so we will be able to focus down to a very small beam size. This beam will also be two to three times more intense. The upgrade should help ensure APS leadership in macromolecular crystallography for many years to come.”

See the full article here.

Argonne is managed by UChicago Argonne, LLC for the U.S. Department of Energy’s Office of Science

The Advanced Photon Source at Argonne National Laboratory is one of five national synchrotron radiation light sources supported by the U.S. Department of Energy’s Office of Science to carry out applied and basic research to understand, predict, and ultimately control matter and energy at the electronic, atomic, and molecular levels, provide the foundations for new energy technologies, and support DOE missions in energy, environment, and national security.

Argonne National Laboratory seeks solutions to pressing national problems in science and technology. The nation’s first national laboratory, Argonne conducts leading-edge basic and applied scientific research in virtually every scientific discipline. Argonne researchers work closely with researchers from hundreds of companies, universities, and federal, state and municipal agencies to help them solve their specific problems, advance America’s scientific leadership and prepare the nation for a better future. With employees from more than 60 nations, Argonne is managed by UChicago Argonne, LLC for the U.S. Department of Energy’s Office of Science.

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From Argonne APS: “A Layered Nanostructure Held Together By DNA”

News from Argonne National Laboratory

March 18, 2014
David Lindley

Dreaming up nanostructures that have desirable optical, electronic, or magnetic properties is one thing. Figuring out how to make them is another. A new strategy uses the binding properties of complementary strands of DNA to attach nanoparticles to each other and builds up a layered thin-film nanostructure through a series of controlled steps. Investigation at the U.S. Department of Energy Office of Science’s Advanced Photon Source has revealed the precise form that the structures adopted, and points to ways of exercising still greater control over the final arrangement.

DNA

The idea of using DNA to hold nanoparticles was devised more than 15 years ago by Chad Mirkin and his research team at Northwestern University. They attached short lengths of single-stranded DNA with a given sequence to some nanoparticles, and then attached DNA with the complementary sequence to others. When the particles were allowed to mix, the “sticky ends” of the DNA hooked up with each other, allowing for reversible aggregation and disaggregation depending on the hybridization properties of the DNA linkers.

Nanoparticles linked by complementary DNA strands form a bcc superlattice when added layer-by-layer to a DNA coated substrate. When the substrate DNA is all one type, the superlattice forms at a different orientation (top row) than if the substrate has both DNA linkers (bottom row). GISAXS scattering patterns (right) and scanning electron micrographs (inset) reveal the superlattice structure. No image credit.

Recently, this DNA “smart glue” has been utilized to assemble nanoparticles into ordered arrangements resembling atomic crystal lattices, but on a larger scale. To date, nanoparticle superlattices have been synthesized in well over 100 crystal forms, including some that have never been observed in nature.

However, these superlattices are typically polycrystalline, and the size, number, and orientation of the crystals within them is generally unpredictable. To be useful as metamaterials, photonic crystals, and the like, single superlattices with consistent size and fixed orientation are needed.

Northwestern researchers and a colleague at Argonne National Laboratory have devised a variation on the DNA-linking procedure that allows a greater degree of control.

The basic elements of the superlattice were gold nanoparticles, each 10 nanometers across. These particles were made in two distinct varieties, one adorned with approximately 60 DNA strands of a certain sequence, while the other carried the complementary sequence.

The researchers built up thin-film superlattices on a silicon substrate that was also coated with DNA strands. In one set of experiments, the substrate DNA was all of one sequence – call it the “B” sequence – and it was first dipped into a suspension of nanoparticles with the complementary “A” sequence.

When the A and B ends connected, the nanoparticles formed a single layer on the substrate. Then the process was repeated with a suspension of the B-type nanoparticles, to form a second layer. The whole cycle was repeated, as many as four more times, to create a multilayer nanoparticle superlattice in the form of a thin film.

Grazing incidence small-angle x-ray scattering (GISAXS) studies carried out at the X-ray Science Division 12-ID-B beamline at the Argonne Advanced Photon Source revealed the symmetry and orientation of the superlattices as they formed. Even after just three half-cycles, the team found that the nanoparticles had arranged themselves into a well-defined, body-centered cubic (bcc) structure, which was maintained as more layers were added.

In a second series of experiments, the researchers seeded the substrate with a mix of both the A and B types of DNA strand. Successive exposure to the two nanoparticle types produced the same bcc superlattice, but with a different vertical orientation. That is, in the first case, the substrate lay on a plane through the lattice containing only one type of nanoparticle, while in the second case, the plane contained an alternating pattern of both types (see the figure).

To get orderly superlattice growth, the researchers had to conduct the process at the right temperature. Too cold, and the nanoparticles would stick to the substrate in an irregular fashion, and remain stuck. Too hot, and the DNA linkages would not hold together.

But in a temperature range of a couple of degrees on either side of about 40° C (just below the temperature at which the DNA sticky ends detach from each other), the nanoparticles were able to continuously link and unlink from each other. Over a period of about an hour per half-cycle, they settled into the bcc superlattice, the most thermodynamically stable arrangement.

GISAXS also revealed that although the substrate forced superlattices into specific vertical alignments, it allowed the nanoparticle crystals to form in any horizontal orientation. The researchers are now exploring the possibility that by patterning the substrate in a suitable way, they can control the orientation of the crystals in both dimensions, increasing the practical value of the technique.

See the full article here.

Argonne is managed by UChicago Argonne, LLC for the U.S. Department of Energy’s Office of Science

The Advanced Photon Source at Argonne National Laboratory is one of five national synchrotron radiation light sources supported by the U.S. Department of Energy’s Office of Science to carry out applied and basic research to understand, predict, and ultimately control matter and energy at the electronic, atomic, and molecular levels, provide the foundations for new energy technologies, and support DOE missions in energy, environment, and national security.

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From Brookhaven Lab: “Understanding the Initiation of Protein Synthesis in Mammals”

Brookhaven Lab

March 18, 2014
Chelsea Whyte

Protein synthesis, the process by which cells generate new proteins, is the most important cellular function, requiring more than 70 percent of the total energy of a cell. The initiation of this process is the most regulated and most critical component, but it is still the least understood.

Messenger RNA (in red) latches closed around a pre-initiation complex, and attaches to transfer RNA (in green), beginning a process of protein synthesis specific to eukaryotes — animals, plants, and fungi.

Research by Ivan Lomakin and Thomas Steitz of Yale University has unlocked the genetic scanning mechanism that begins this crucial piece of cell machinery.

They determined the structures of three complexes of the ribosome, a complex molecular machine that links together amino acids to form proteins according to an order specified by messenger RNA. These three structures represent distinct steps in protein translation in mammals – the recruitment and scanning of mRNA, the selection of initiator tRNA, and the joining of large and small ribosomal subunits.

“Any small defect or disruption in the protein synthesis process can cause abnormalities or disease,” said Lomakin. “Understanding this process is critical for understanding how human life comes to be, and how some over-expressions or abnormalities in the initiation of protein synthesis may be connected to cancer or Alzheimer’s or other diseases.”

Using x-ray crystallography on ribosomal subunits purified from rabbit cells, they were able to determine the positions and roles of the different pieces of cellular machinery, a bit like creating a playbook for a football game. They found that the tRNA and mRNA compete for position at the P site – one of three key sites on a ribosome – where short chains of amino acids are linked to form proteins.

“Now, we have a low resolution structure, so we can’t yet talk about atomic details of the mechanism,” said Lomakin. “The next important step is to get higher resolution images. And we can change organisms to see if they behave differently, so we’re working on the structure of human ribosome initiation complexes, too.”

For this higher resolution, Lomakin and his collaborators will use the National Synchrotron Light Source II, a new state-of-the-art light source that will begin early science at Brookhaven National Laboratory in 2014. “Our hope is to be able to look at very weak diffraction to get higher resolution structures of these important cellular mechanisms.”

NSLS-II at Brookhaven Lab

See the full article here.

One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. The Laboratory’s almost 3,000 scientists, engineers, and support staff are joined each year by more than 5,000 visiting researchers from around the world.Brookhaven is operated and managed for DOE’s Office of Science by Brookhaven Science Associates, a limited-liability company founded by Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization.

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From Berkeley Lab: “Bright Future for Protein Nanoprobes”

Berkeley Lab

March 17, 2014
Lynn Yarris (510) 486-5375 lcyarris@lbl.gov

The term a “brighter future” might be a cliché, but in the case of ultra-small probes for lighting up individual proteins, it is now most appropriate. Researchers at the U.S. Department of Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) have discovered surprising new rules for creating ultra-bright light-emitting crystals that are less than 10 nanometers in diameter. These ultra-tiny but ultra-bright nanoprobes should be a big asset for biological imaging, especially deep-tissue optical imaging of neurons in the brain.

Working at the[Berkeley Lab’s] Molecular Foundry, a DOE national nanoscience center hosted at Berkeley Lab, a multidisciplinary team of researchers led by James Schuck and Bruce Cohen, both with Berkeley Lab’s Materials Sciences Division, used advanced single-particle characterization and theoretical modeling to study what are known as “upconverting nanoparticles” or UCNPs. Upconversion is the process by which a molecule absorbs two or more photons at a lower energy and emits them at higher energies. The research team determined that the rules governing the design of UCNP probes for ensembles of molecules do not apply to UCNP probes designed for single-molecules.

“The widely accepted conventional wisdom for designing bright UCNPs has been that you want to use a high concentration of sensitizer ions and a relatively small concentration of emitter ions, since too many emitters will result in self-quenching that leads to lower brightness, says Schuck, who directs the Molecular Foundry’s Imaging and Manipulation of Nanostructures Facility. “Our results show that under the higher excitation powers used for imaging single particles, emitter concentrations should be as high as possible without compromising the structure of the nanocrystal, while sensitizer content can potentially be eliminated.”

From left Bruce Cohen, Emory Chan, Dan Gargas and Jim Schuck led a study at the Molecular Foundry to develop ultra-small, ultra-bright nanoprobes that should be a big asset for biological imaging, especially imaging neurons in the brain. (Photo by Roy Kaltschmidt)

Schuck and Cohen are the corresponding authors of a paper describing this research in Nature Nanotechnology. The paper is titled Engineering bright sub-10-nm upconverting nanocrystals for single-molecule imaging. Co-authors are Daniel Gargas, Emory Chan, Alexis Ostrowski, Shaul Aloni, Virginia Altoe, Edward Barnard, Babak Sanii, Jeffrey Urban and Delia Milliron.

Proteins are one of the fundamental building blocks of biology. The cells that make up tissues and organs are constructed from assemblies of proteins interacting with other biomolecules, while other proteins control nearly every chemical process inside a cell. Studying the location, assembly, and movement of specific proteins is essential for understanding how cells function and what goes wrong in diseased cells. Scientists often study proteins within cells by labeling them with light-emitting probes, but finding probes that are bright enough for imaging but not so large as to disrupt the protein’s function has been a challenge. Fluorescent organic dye molecules and semiconductor quantum dots meet the size requirements but impose other limitations.

“Organic dyes and quantum dots will blink, meaning they randomly turn on and off, which is quite problematic for single-molecule imaging, and will photobleach, turn-off permanently, usually after less than 10 seconds under most imaging conditions,” Schuck says.

Five years ago, Cohen and Schuck and their colleagues at the Molecular Foundry synthesized and imaged single UCNPs made from nanocrystals of sodium yttrium fluoride (NaYF4) doped with trace amounts of the lanthanide elements ytterbium, for the sensitizer ions, and erbium, for the emitter ions. These UCNPs were able to upconvert near-infrared photons into green or red visible light, and their photostability makes them potentially ideal luminescent probes for single-molecule imaging.

“Cells don’t naturally contain lanthanides, so they don’t upconvert light at all, which means we can image without any measurable background,” Cohen says. “And we can excite with near-infrared light, which is a lot less damaging to cells than visible or ultraviolet light. These are great properties, but to make our UCNPs more compatible with cellular imaging, we had to develop new synthetic methods to make them smaller.”

However, when Foundry scientists shrunk UCNP size, following the conventional design rules, they found that loss of brightness became a major issue. UCNPs smaller than 10 nanometers were no longer bright enough for single molecule imaging. This prompted the new study, which showed that factors known to increase brightness in bulk experiments lose importance at higher excitation powers and that, paradoxically, the brightest probes under single-molecule excitation are barely luminescent at the ensemble level.

The Molecular Foundry is one of five DOE Nanoscale Science Research Centers (NSRCs) dedicated to interdisciplinary research at the nanoscale. The Foundry is hosted at Berkeley Lab. (Photo by Roy Kaltschmidt, Berkeley Lab)

“This discovery came about really as a consequence of the multidisciplinary collaborative environment at the Molecular Foundry,” says Daniel Gargas, co-lead author of the Nature Nanotechnology paper. “By utilizing our daily contact and friendships with scientists throughout the Foundry, we were able to perform highly advanced research on nanoscale materials that included the study of single-molecule photophysics, the ability to synthesize ultra-small upconverting nanocrystals of almost any composition, and the advanced modeling/simulation of UCNP optical properties. There aren’t many facilities in the world that can match this collaborative atmosphere with such high levels of scientific characterization.”

UCNPs make use of sensitizer ions, such as ytterbium, with relatively large photon absorption cross-sections, to absorb incoming light and transfer this absorbed energy to emitter ions, such as erbium, which luminesce. The original lanthanide-doped UCNPs contained 20-percent ytterbium and 2-percent erbium, which were believed to be the optimal concentrations for brightness in both bulk and nanocrystals. However, the new Molecular Foundry study showed that for UCNPs smaller than 10 nanometers, the erbium concentration could be raised to 20-percent and the ytterbium concentration could be reduced to 2-percent, or even eliminated for UCNPs approaching five nanometers.

”People often assume that particles that are the brightest at low powers will also be the brightest at high powers, but we found our ultra-small UCNPs to be a classic tortoise-and-hare example,” says Emory Chan, the other co-lead author of the Nature Nanotechnology paper. “UCNPs heavily doped with erbium start slowly out of the gate, being incredibly dim at low powers, but by the time the laser intensity is cranked up to high power, they have passed up the conventionally doped UCNPs that are the high-flyers at low powers.”

Chan’s computer models predict that the new rules are universal for lanthanide-doped nanocrystal hosts and he is now using the Foundry’s WANDA robot (Workstation for Automated Nanomaterial Discovery and Analysis), which he developed along with co-author Delia Milliron, to create and screen for the best UCNP compositions based on different operation/application considerations and criteria.

In the course of discovering the new rules for designing ultra-small UCNPs, the research team also discovered that complex levels of heterogeneity exist within the emission spectra of these UCNPs. This suggests that emissions from the UCNPs may be originating from only a small subset of the total emitters.

“Future studies may determine how to engineer particles consisting of only these super-emitters resulting in even brighter emissions from ultra-small UCNPs,” Gargas says.

This research was supported by the DOE Office of Science.

See the full article here.

A U.S. Department of Energy National Laboratory Operated by the University of California

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From Livermore Lab: “New X-ray analysis method opens the door to researching an elusive class of proteins”

Lawrence Livermore National Laboratory

02/13/2014
Stephen P Wampler, LLNL, (925) 423-3107, wampler1@llnl.gov

An international team of researchers has demonstrated a new method for studying the structure of proteins that could lead to important advances in biology and other fields.

Beam line scientist Marc Messerschmidt loads a sample holder that supports two-dimensional protein crystals into the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. He co-authored an International Union of Crystallography Journal article on a new method of studying proteins.

For the first time, protein crystals have been studied in two dimensions at room temperature with X-rays, using a new technique that could open the door for scientists to learn more about an important class of proteins that constitute about one-third of all human proteins. The scientists report their findings about this X-ray diffraction method in the March edition of the International Union of Crystallography Journal.

“Our work demonstrates for the first time that two-dimensional protein X-ray crystallography is a potential method for obtaining protein structure information,” said Matthias Frank, a physicist at the Department of Energy’s (DOE) Lawrence Livermore National Laboratory (LLNL).

Frank co-led a team of two dozen scientists with crystallographer James Evans of Pacific Northwest National Laboratory (PNNL) that used the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory in Menlo Park, Calif. The SLAC machine, an X-ray Free Electron Laser (XFEL), is one of two such machines in the world — along with Japan’s SACLA — that can produce hard X-rays.

Using the predominant method for analyzing protein structures, synchrotron light sources, researchers who take snapshots of proteins must work in three dimensions and need a billion copies of the same protein all stacked up on each other to get a better image of what the protein looks like.

In effect, synchrotron machines require crystals to be grown in three dimensions to distribute the radiation damage from the X-rays over many molecules, but that makes it difficult to study about one-third of all human proteins, called membrane proteins, because crystals of these proteins are hard to grow in three dimensions.

Now with the LCLS XFEL machine, scientists can use exceptionally bright and fast X-rays to take a picture that rivals current methods, with a sheet of proteins only one layer thick.

“We can do this because the X-ray pulses from the LCLS are so bright and so fast that we can produce a measurable X-ray diffraction signal before the sample is destroyed. This type of two-dimensional measurement is not possible at synchrotron light sources because the sample is destroyed by radiation before enough of the signal can be obtained,” Frank said.

The membrane proteins that can now be studied more easily using the LCLS machine are often embedded in the boundaries of cells and perform a number of vital cell functions, including nutrient uptake, transport and signaling.

They are important not only because they represent about one-third of all human proteins, but also because they constitute about two-thirds of all current pharmaceutical drug targets for fighting diseases and other human-related health issues.

“The structures of the proteins tell us about their function, which is why it’s important to better understand their structure,” according to Frank.

“We believe that ultimately the two-dimensional X-ray crystallography performed at room temperature may someday allow us to observe molecules in action, or to make what some call ‘molecular movies.’ This would permit us to better understand the proteins’ functions and how to control them by drugs,” he continued.

In 2012, scientists showed they could use an XFEL on a crystal of proteins 10 to 20 sheets thick. Frank, Evans and their team aimed at reducing the number of layers further.

The team developed a way to create one-sheet-thick layers of two different proteins — a soluble protein called streptravidin that is a commonly used molecule in biology for binding substances and bacteriorhdopsin, a membrane protein isolated from bacteria living in salt ponds. The structures of both proteins are well understood, so the proteins served as model systems.

The team measured the resolution of the two proteins at 8.5 angstroms (a size that would fit in the period at the end of a sentence more than 500,000 times). In subsequent experiments, they have already obtained better resolutions and eventually, the scientists would like to reach a resolution of 2 angstroms, a level at which individual atoms can be distinguished.

In addition to Frank, other members of the LLNL multidisciplinary team that assisted in conducting the three-day May 2012 experiments were postdoctoral researcher and physical chemist Mark Hunter, chemist W. Henry Benner, physicist Stefan Hau-Riege, postdoctoral researchers and physicists Tommaso Pardini and Alexander Graf (who has since left LLNL), and biologists Brent Segelke and Matthew Coleman.

Among the institutions that participated in the research besides LLNL and PNNL were the University of California, Davis, Arizona State University, the Centre for Free Electron Laser Science based in Hamburg, Germany; the Paul Scherrer Institute in Villigen, Switzerland; and the LCLS at the SLAC National Accelerator Laboratory.

Since 2005, LLNL has conducted proof-of-principle experiments for using X-ray free electron lasers for biological imaging and structural biology. Those experiments were led initially by former LLNL physicist Henry Chapman, who is now a director at the Centre for Free Electron Laser Science in Hamburg.

The paper is published on the Web in the International Union of Crystallography Journal.

See the full article here.

Operated by Lawrence Livermore National Security, LLC, for the Department of Energy’s National Nuclear Security
Administration



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