Click chemistry with copper -- a biocompatible version

Berkeley Lab researchers have found a way to make copper-catalyzed click chemistry biocompatible. By adding a ligand that minimizes the toxicity of copper but still allows it to catalyze the click chemistry reaction, the researchers can safely use their reaction in living cells.

Biomolecular imaging can reveal a great deal of information about the inner workings of cells and one of the most attractive targets for imaging are glycans – sugars that are ubiquitous to living organisms and abundant on cell surfaces. Imaging a glycan requires that it be tagged or labeled. One of the best techniques for doing this is a technique called click chemistry. The original version of click chemistry could only be used on cells in vitro, not in living organisms, because the technique involved catalysis with copper, which is toxic at high micromolar concentrations. A copper-free version of click chemistry that can safely be used in living organisms is available, but it is not always optimal in terms of reaction kinetics and target specificity. Now, a variation of click chemistry has been introduced that retains the copper catalyst of the original reaction - along with its speed and specificity – but is safe for cells in vivo.

Researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab), in collaboration with researchers at the Albert Einstein College of Medicine at Yeshiva University in New York, have found a way to make copper-catalyzed click chemistry biocompatible. By adding a ligand that minimizes the toxicity of copper but still allows it to catalyze the click chemistry reaction, the researchers can safely use their reaction in living organisms. Compared to the copper-free click chemistry reaction, which can take up to an hour, the ligand-accelerated copper-catalyzed click chemistry reaction can achieve effective labeling within 3-5 minutes. The presence of the copper catalyst also enables this new formulation of click chemistry to be more target-specific with fewer background side reactions.

"The discovery of this new accelerating ligand for copper-catalyzed click chemistry should provide an effective complimentary tool to copper-free click chemistry," says Yi Liu, a chemist with Berkeley Lab's Molecular Foundry and the co-leader of this research with Peng Wu, of the Albert Einstein College of Medicine.

"While copper-free click chemistry may have advantages for whole animal imaging experiments such as imaging in mice," Liu says, "our ligand-accelerated copper reaction is better suited for enriching glycoproteins for their identification."

The ligand-accelerated copper-catalyzed reaction was used to label glycans in recombinant glycoproteins, glycoproteins in cell lysates, glycoproteins on live cell surfaces, and glycoconjugates in live zebrafish embryos. Because a zebrafish embryo is transparent in the first 24 hours of its development, it allows labeled glycans to be detected via molecular imaging techniques, making it a highly useful model for developmental biology studies.

"Based on our results," says Peng Wu, "we believe that ligand-accelerated copper-catalyzed click chemistry represents a powerful and highly adaptive bioconjugation tool that holds great promise for further improvement with the discovery of more versatile catalyst systems."

Click chemistry, which was introduced in 2002 by the Nobel laureate chemist Barry Sharpless of the Scripps Research Institute, utilizes a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction that makes it possible for certain chemical building blocks to "click" together in an irreversible linkage, analogous to the snapping together of Lego blocks. While the technique immediately proved valuable for attaching small molecular probes to various biomolecules in a test tube or on fixed cells, it could not be used for biomolecule labeling in live cells or organisms because of the copper catalyst.

In 2007, Carolyn Bertozzi, a chemist who holds joint appointments with Berkeley Lab, the University of California (UC) Berkeley, and the Howard Hughes Medical Institute, led a research effort that produced a copper-free version of click chemistry. In this version, glycans were metabolically labeled with azides - a functional group featuring three nitrogen atoms - via reactions that were carried out through the use of cyclooctyne reagents that required no copper catalyst. With their latest reagent, biarylazacyclooctynone (BARAC), Bertozzi and her group have provided a copper-free click chemistry technique that delivers relatively fast reaction kinetics and the bioorthogonality needed for biomolecule labeling. However, the technique can only be used on biomolecules that can be tagged with azides.

"Our bio-benign ligand-accelerated copper-catalyzed click chemistry reaction liberates bioconjugation from the limitation where ligations could only be accomplished with azide-tagged biomolecules," Liu says. "Now terminal alkyne residues can also be incorporated into biomolecules and detected in vivo."

Provided by Lawrence Berkeley National Laboratory (news : web)

MS research: Myelin influences how brain cells send signals

The development of a new cell-culture system that mimics how specific nerve cell fibers in the brain become coated with protective myelin opens up new avenues of research about multiple sclerosis. Initial findings suggest that myelin regulates a key protein involved in sending long-distance signals.

Multiple sclerosis (MS) is an autoimmune disease characterized by damage to the myelin sheath surrounding nerve fibers. The cause remains unknown, and it is a chronic illness affecting the central nervous system that has no cure.

MS has long been considered a disease of white matter, a reference to the white-colored bundles of myelin-coated axons that project from the main body of a brain cell. But researchers have discovered that the condition also affects myelinated axons scattered in gray matter that contains main bodies of brain cells, and specifically the hippocampus region, which is important for learning and memory.

Up to half of MS patients suffer cognitive deficits in addition to physical symptoms. Researchers suspect that cognitive problems are caused by abnormal electrical activities of the demyelinated axons extending from hippocampal cells, but until now have not been able to test myelin's role in this part of the brain.

Ohio State University researchers have created a system in which two types of cells interact in a dish as they do in nature: neurons from the hippocampus and other brain cells, called oligodendrocytes, whose role is to wrap myelin around the axons.

Now that the researchers can study how myelination is switched on and off for hippocampal neurons, they also can see how myelin does more than provide insulation – it also has a role in controlling nerve impulses traveling between distant parts of the nervous system. Identifying this mechanism when myelin is present will help improve understanding of what happens when axons in this critical area of the brain lose myelin as a result of MS, researchers say.

So far, the scientists have used the system to show that myelin regulates the placement and activity of a key protein, called a Kv1.2 voltage-gated potassium channel, that is needed to maintain ideal conditions for the effective transmission of electrical signals along these hippocampal axons.

"This channel is important because it is what leads to electrical activity and how neurons communicate with each other downstream," said Chen Gu, assistant professor of neuroscience at Ohio State and lead author of the study. "If that process is disrupted by demyelination, disease symptoms may occur."

The study appears in the current (July 22, 2011) issue of the Journal of Biological Chemistry.

To create the cell culture system, the researchers began with hippocampus neurons from a rodent brain – a cell type that Gu has worked with for years. In culture, these cells can grow and develop dendrites – other branch-like projections off of neurons – and axons as well as generate electrical activity and synaptic connections, the same events that occur in the brain.

The researchers then added oligodendrocytes, along with some of their precursor cells, to the same dish as the neurons. And eventually, after maturing, these oligodendrocytes began to wrap myelin around the axons of the hippocampal neurons.

This system takes about five weeks to create, but the trickiest part, Gu said, was developing the proper solution for this culture so that both kinds of cells would behave as nature intended.

"In the end, the composition of the culture medium is basically half from a solution that supports the neurons and half from a medium in which the oligodendrocytes function well. We know that all the cells were happy because we got myelin," said Gu, also an investigator in Ohio State's Center for Molecular Neurobiology.

With the system established, they then turned to experimentation to test the effects of the myelin's presence on these specific brain cells.

Nerve cells send their signals encoded in electrical impulses over long distances. Concerted actions of various ion channels are required for properly generating these nerve impulses. Potassium channels are involved at the late phase in an impulse, and its role is to return a nerve cell to a resting state after the impulse has passed through it and gear up for the next one. The Kv1.2 ion channel helps ensure that this process works smoothly.

By experimentally manipulating signal conditions with the new co-culture system, Gu and his colleague were able to establish part of the sequence of events required for myelinated hippocampal neurons to effectively get their signals to their targets. Starting with a protein known to be produced by myelin and axons, called TAG-1, a cell adhesion molecule, they traced a series of chemical reactions indicating that myelin on the hippocampal axons was controlling the placement and activity of the Kv1.2 ion channel.

"The analysis allowed us to see the signaling pathways involving myelin's regulation of the Kv1.2 channel's placement along the axon as well as fine-tuning of the channel's activity," Gu said.

When MS demyelinates these axons, the affected nerve cells don't get the message to rest, and subsequently can't prepare adequately to receive and transmit the next signal that comes along.

"This means a nerve impulse will have a hard time traveling through the demyelinated region," Gu said. "This shows that the ion channel is probably involved in the downstream disease progression of MS."

Gu envisions many additional uses for the new co-culture system, including additional studies of how myelin affects the behavior of other channels, proteins and molecules that function within axons, as well as to screen the effects of experimental drugs on these myelinated cells.

This work was supported by a Career Transition Fellowship Award from the National Multiple Sclerosis Society and a grant from the National Institute for Neurological Disorders and Stroke.

Provided by The Ohio State University (news : web)

Juvenile diarrhea virus analyzed

Rice University scientists have defined the structure -- down to the atomic level -- of a virus that causes juvenile diarrhea. The research may help direct efforts to develop medications that block the virus before it becomes infectious.

The new paper by Professor Yizhi Jane Tao, postdoctoral researcher Jinhui Dong and their colleagues was published in today's online edition of the Proceedings of the National Academy of Sciences.

Tao's Rice lab specializes in gleaning fine details of viral structures through X-ray crystallography and computer analysis of the complex molecules, ultimately pinpointing the location of every atom. That helps researchers see microscopic features on a virus, like the spot that allows it to bind to a cell or sites that are recognized by neutralization antibodies.

Among four small RNA viruses that typically infect people and animals, Tao said, astrovirus was the only one whose atomic structure was not yet known. First visualized through electron microscopy in 1975, it became clear in subsequent studies that the virus played a role in juvenile -- and sometimes adult -- outbreaks of diarrhea, as the second leading cause after rotavirus. Passed orally, most often through fecal matter, the illness is more inconvenient than dangerous, but if left untreated, children can become dehydrated.

The virus works its foul magic in humans' lower intestines, but to get there it has to run a gauntlet through the digestive tract and avoid proteases, part of the human immune system whose job is to destroy it. (Though one, trypsin, actually plays a role in activating astrovirus, she said.) When the astrovirus finds a target and viral RNA is let loose inside human cells, virus replication starts. If the host's immune system does not do a good enough job in removing the viruses, the malady will run its uncomfortable course in a couple of days.

 

Rice University postdoctoral researcher Jinhui Dong mounts a protein crystal to an X-ray machine to collect diffraction data. Credit: Jeff Fitlow/Rice University

Astrovirus bears a strong resemblance to the virus that causes hepatitis E (HEV). Tao, an associate professor of biochemistry and cell biology, said she decided to investigate astrovirus after completing a similar study of HEV two years ago. "I was thinking there's some connection between those viruses," she said. "Based on that assumption, we started to make constructs to see if we could produce, to start with, the surface spike on the viral capsid."

The capsid is a hard shell 33 nanometers wide that contains and protects its RNA. It has 30 even tinier spikes projecting from the surface, and each of those spikes may have a receptor-binding site.

Once the atomic structure of the spike was known, finding the receptor site took detective work that involved comparing genomic sequences of eight variants of astrovirus to find which were the best conserved. "Among those eight serotypes, we figured there must be a common receptor, and that should be conserved on the surface," said Dong, the paper's lead author.

In looking for the common receptor, the team found a shallow pocket in the spike that became a prime suspect for receptor binding.

The researchers also discovered the astrovirus may have a sweet tooth. "The size of the pockets suggests that it would most likely bind to sugar molecules, like disaccharides or trisaccharides," Tao said. "It may be that the virus binds to the sugar molecule and that helps it bind to the surface of a target cell."

Finally, the team also determined astrovirus resembles another of the four types of RNA-based viruses, calicivirus, although more remotely than HEV. They suspect astrovirus may be a hybrid, with parts derived from both HEV and calicivirus. "Clearly, these three are related somehow. It's an interesting point, but we can't determine that relationship based on what we know right now."

What researchers can do is begin to develop a vaccine or antiviral drug that will block astrovirus. "There's already a phase II vaccine (in trials) for HEV, so that gives us hope," Dong said.

"We will certainly work with other labs to identify compounds that can bind to this potential pocket," Tao said. "We can do this computationally. We can screen 50,000 compounds, for example, to see which may bind to the protein with high affinity. Then we can start the optimization procedure."

Provided by Rice University (news : web)

Scientists describe the birth of a protein

Yale researchers for the first time have captured the chemical reaction that occurs when a protein is created — one of life's most basic processes.

Proteins are synthesized within cells by ribosomes, which take genetic information encoded by DNA and delivered by RNA and convert it into proteins, which carry out the business of life. But the key moment in the process -- when chemical bonds of the proteins form a chain of amino acids called peptides -- had never been described.

The image here represents the transition state for this reaction when a peptide is formed in a growing peptide chain.

The work is published online in the July 17 issue of the journal Nature. The lead author of the paper was David Hiller, a postdoctoral researcher working in the lab of senior author Scott Strobel, the Henry Ford II Professor of Molecular Biophysics and Biochemistry and professor of chemistry.

More information: The full paper is available on Nature website: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10248.html

Provided by Yale University (news : web)

Antibacterial stainless steel created

Materials scientists at the University of Birmingham have devised a way of making stainless steel surfaces resistant to bacteria in a project funded by the Engineering and Physical Sciences Research Council which culminated this week.

By introducing silver or copper into the steel surface (rather than coating it on to the surface), the researchers have developed a technique that not only kills bacteria but is very hard and resistant to wear and tear during cleaning. 

Bacteria resistant surfaces could be used in hospitals to prevent the spread of superbug infections on stainless steels surfaces, as well as in medical equipment, for example, instruments and implants.  They would also be of use to the food industry and in domestic kitchens. 

The team has developed a novel surface alloying technology using Active Screen Plasma (ASP) with a purpose designed composite or hybrid metal screen.  The combined sputtering, back-deposition and diffusion allows the introduction of silver into a stainless steel surface, along with nitrogen and carbon.  The silver acts as the bacteria killing agent and the nitrogen and carbon make the stainless steel much harder and durable. 

The researchers replicated the cleaning process for medical instruments in hospitals.  After cleaning the treated instruments 120 times they found that the antibacterial properties of the stainless steel were still intact and the surface still resistant to wear. 

Hanshan Dong, Professor of Surface Engineering at the University of Birmingham and lead investigator, said: ‘Previous attempts to make stainless steel resistant to bacteria have not been successful as these have involved coatings which are too soft and not hard-wearing.  Thin antibacterial coatings can be easily worn down when interacting with other surfaces, which leads to a low durability of the antibacterial surface.  Our technique means that we avoid coating the surface, instead we modify the top layers of the surface.’

Professor Dong’s team are confident that this technique could be used in the manufacturing of stainless steel products as they are already able to surface engineer items of up to two metres x two metres in the laboratory.

Provided by University of Birmingham