Power of Protein Crystal Structures
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BNL Newsroom | Structure Helps Yield Drug "Hypersensitivity" Tests for Patients

BNL Newsroom | Structure Helps Yield Drug "Hypersensitivity" Tests for Patients | Power of Protein Crystal Structures | Scoop.it
Researchers are studying an antiviral drug known to cause hypersensitivity in patients who carry a particular gene.

 

From a patient's point of view, one of the unsettling things about taking a new drug is the possibility of unwelcome side effects or worse, dangerous allergic reactions. As drugs are being developed and then enter clinical trials, these issues play a huge role in the process, increasing costs and sometimes determining whether a drug will get to market at all.

One type of severe reaction is "hypersensitivity," in which the immune system over-reacts to a substance that is foreign but not infectious, producing symptoms that can be mild (such as rashes) to severe (organ failure, even death). In this study, researchers studied an antiviral drug known to cause hypersensitivity in patients who carry a particular gene. Using x-rays at Brookhaven Lab's National Synchrotron Light Source(NSLS), they were able to "see" how at the molecular level, the drug binds to the protein created from the gene, triggering the immune response. Their work has produced new ways to predict whether a drug is likely to induce a gene-based allergic reaction.

The drug they studied is abacavir, an antiviral used to treat HIV and AIDS. About eight percent of patients who take it experience hypersensitivity; out of these patients, the majority carry a particular gene known as HLA-B*57:01. It is a member of the "human leukocyte antigen" (HLA) gene family, which contains a large number of genes involved in the body's immune response.

"Many drug hypersensitivity reactions are HLA-linked, meaning that they will occur much more often or even exclusively in individuals who have certain variants of the HLA gene," said the study's lead scientist, Bjoern Peters from the La Jolla Institute for Allergy & Immunology in San Diego, CA. "The present system of clinical trials is very powerful in identifying side effects that occur in many people. However, HLA-linked hypersensitivity has been a really big problem that often doesn't surface until after the drug is approved and taken by thousands of people."

Scientists have proposed a few mechanisms by which abacavir may bind to HLA-B*57:01, but until this study none had been proven experimentally. Peters and his group favored the idea that abacavir can bind within the HLA-B*57:01 "peptide binding groove," a cleft on the gene in which other molecules can dock. This would interrupt the usual activity that occurs at the site, namely binding to certain peptides (short protein-like chains) and presenting them to T-cells, a type of white blood cell that is a key part of the immune system. Binding to abacavir, therefore, would alter how the gene communicates with receptors on the T-cell surfaces.

To verify this, Peters and his group first confirmed abacavir's tendency to bind to the protein generated from this gene (versus other genes in the HLA family) via a series of tests. They then approached a collaborator, David Ostrov from the University of Florida, to help grow crystals (a crystal is a repeating pattern of the same molecular unit) that consist of HLA-B*57:01 bound to a certain peptide (they call it pep-V) in the presence of abacavir. That particular peptide was chosen to maximize the chance of getting a good look at how abacavir is bound.

Ostrov then sent his crystals to Jean Jakoncic at the National Institutes of Health's National Institute of General Medical Sciences East Coast Structural Biology Facility, located at NSLS. At beamline X6A, the group used a technique called x-ray crystallography to "solve" the molecular structure of the abacavir/peptide/HLA-B*57:01 complex. X-rays aimed at the crystal produced an intricate diffraction pattern, from which the researchers could work backward and deduce what the complex looked like.

They represent the structure using a figure known as a ribbon diagram.

They discovered that abacavir binds to a certain pocket within the binding groove and, in the process, forms weak bonds to both pep-V and HLA-B*57:01. Further tests confirmed that the presence of abacavir does cause the gene to present a different set of peptides to the T-cell receptors.

"We found that certain drugs can alter which peptides specific HLA molecules show to the immune system," said Peters. "Abacavir causes a self-peptide, derived from a human protein, to be shown to the immune system by the HLA-B*57:01. This peptide would otherwise never be seen by the immune system." Having not previously recognized the peptide, Peters said, the immune system mistakes it for being derived from an invader and launches an attack. This immune attack results in drug hypersensitivity.

Peters and his team have established assays (tests) that can be applied to test drug compounds to determine if a specific HLA variant reaction occurs. "You wouldn't have to wait until after the drug is introduced and thousands of people are potentially affected," he said, explaining the testing could be done in human blood samples. "This type of testing could be done before human subjects are exposed to the drug, and could lead to the design of drug types that do not have this effect, or – if this is not possible – to ensure that only individuals who do not have this HLA variant are given the drug, as is now done for abacavir."

Peters' collaborators on this study include scientists from the University of Florida College of Medicine, the University of Michigan, the National Center for Toxicological Research, the University of Virginia, and the University of Copenhagen. The results are published in the June 19, 2012 edition of the Proceedings of the National Academy of Sciences.

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Serotonin Receptors Offer Clues to New Antidepressants: Scientific American

Serotonin Receptors Offer Clues to New Antidepressants: Scientific American | Power of Protein Crystal Structures | Scoop.it
Long-sought specificity on the shapes of serotonin binding sites could aid in the discovery of new drugs to combat depression as well as in the study of consciousness

 

Researchers have deciphered the molecular structures of two of the brain's crucial lock-and-key mechanisms. The two molecules are receptors for the natural neurotransmitter serotonin — which regulates activities such as sleep, appetite and mood — and could provide targets for future drugs to combatdepression, migraines or obesity. 

“This is huge,” says Bryan Roth, a neuropharmacologist at the University of North Carolina Chapel Hill Medical School, and a co-author of the two studies published in Science today. “Before this there was no crystal structure for any serotonin receptor. A lot of what was theoretical is now known with a great degree of certainty,” he says.

Scientists have been trying to decipher serotonin receptors for years. Armed with information on the atomic level, they might now be able to make breakthroughs in drug discovery and in understanding how the physical structures of the brain produce consciousness, says Roth.

Christoph Anacker, a neuropharmacologist at King's College London, agrees that the findings are important for drug discovery. “These receptors are involved in so many conditions, especially depression, and knowing the molecular structures will help to develop more specific drugs and avoid the expression of undesired side effects.”

Chemical messengers
There are 14 different known serotonin receptors. The molecules lie on the outer membranes of nerve cells; when drugs or neurotransmitters lock into the receptors from outside the cell, they trigger the release of other chemicals inside the cell. Those chemicals — which can be different depending on what drug or neurotransmitter has triggered them — activate further hormones and metabolites, producing signaling cascades that are ultimately responsible for many aspects of the way we feel, perceive and behave.

Some drugs bind at more than one receptor, setting off not-fully-understood reactions that can produce unwanted side effects. To avoid this, researchers want to fine-tune drugs so that they activate only the desired signaling pathway.

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Brighter, Smaller, Faster - X-ray crystallography enters its second century

Brighter, Smaller, Faster - X-ray crystallography enters its second century | Power of Protein Crystal Structures | Scoop.it

It was just over a century ago that William Lawrence Bragg and his father, William Henry Bragg, kick-started the science of X-ray crystallography in a talk at the Cambridge Philosophical Society on November 11, 1912. Since then, thousands upon thousands of structures have been solved, from table salt and diamond to RNA polymerase and the ribosome.

 

Back in the Braggs’ time, crystals were analyzed using laboratory X-ray tubes, which are relatively weak sources of continuous, noncoherent light—that is, light that travels in all directions, like lamplight. In the 1970s, researchers started using synchrotron particle accelerators, which can shoot partially focused (i.e., relatively coherent), highly intense X-ray radiation at a crystal. According to Jianwei Miao, a professor of physics and astronomy at the University of California, Los Angeles, a synchrotron can produce at least nine orders of magnitude more photons per second than a lab X-ray source, producing higher-quality diffraction data from smaller crystals.

 

Since 2009, a select number of researchers have had another option. The Linac [linear accelerator] Coherent Light Source (LCLS) at Stanford University and the SACLA (SPring-8 Angstrom Compact Free Electron Laser) in Japan are the world’s first “hard” X-ray free-electron lasers (XFELs), capable of producing light some billion times more brilliant than that from a synchrotron, and colliding it with tiny crystals in pulses just femtoseconds long. That’s like taking all the sunlight that hits the Earth and focusing it into one square millimeter, explains Janos Hajdu, a professor of molecular biophysics at Uppsala University in Sweden. Under such intense irradiation, the sample is destroyed almost instantaneously—but not before it diffracts, producing a weak but detectable signal.

 

As X-ray intensity increased, the crystal size needed to solve a structure has decreased, from about 1 mm or more for an X-ray tube, to 100–200 μm for a synchrotron, to as small as 200 nm for an X-ray laser, says Sébastien Boutet, a staff scientist at LCLS. That’s a boon for structural biologists, because growing large crystals has been a perennial source of torment. That synchrotrons and XFELs can use smaller crystals, which are easier and faster to grow, has improved matters—but the ultimate goal is to get rid of crystals altogether: to image individual molecules, molecular complexes, or viruses. That would represent a huge advance for scientists; not all proteins crystallize, and those that do sometimes adopt conformations that are not representative of their native forms in vivo.

 

Researchers have not reached that crystal-free point yet, at least not with individual proteins, but they have made significant leaps.


Via Dr. Stefan Gruenwald
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Shedding light on chemistry with a biological twist

Shedding light on chemistry with a biological twist | Power of Protein Crystal Structures | Scoop.it
(Phys.org) —Many of life's processes rely on light to trigger a chemical change. Photosynthesis, vision, the movement of light-seeking or light-avoiding bacteria, for instance, all exploit photochemistry.
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A tribute to G. N. Ramachandran - A Golden Jubilee

A tribute to G. N. Ramachandran - A Golden Jubilee | Power of Protein Crystal Structures | Scoop.it

http://www.iucr.org/news/newsletter/volume-12/number-3/50-years-of-collagen-triple-helix

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Study: Protein structure discovery could lead to better treatments for HIV, early aging

Study: Protein structure discovery could lead to better treatments for HIV, early aging | Power of Protein Crystal Structures | Scoop.it
Researchers at the University of Virginia School of Medicine have determined the molecular structure of a protein whose mutations have been linked to several early aging diseases, and side effects for common HIV and AIDS medications.
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New vaccine-design approach targets viruses such as HIV

New vaccine-design approach targets viruses such as HIV | Power of Protein Crystal Structures | Scoop.it
Scientists have unveiled a new technique for vaccine design that could be particularly useful against HIV and other fast-changing viruses.

 

A team led by scientists from The Scripps Research Institute (TSRI) and the International AIDS Vaccine Initiative (IAVI) has unveiled a new technique for vaccine design that could be particularly useful against HIV and other fast-changing viruses.

The report, which appears March 28, 2013, in Science Express, the early online edition of the journal Science, offers a step toward solving what has been one of the central problems of modern vaccine design: how to stimulate the immune system to produce the right kind of antibody response to protect against a wide range of viral strains. The researchers demonstrated their new technique by engineering an immunogen (substance that induces immunity) that has promise to reliably initiate an otherwise rare response effective against many types of HIV.

"We're hoping to test this immunogen soon in mice engineered to produce human antibodies, and eventually in humans," said team leader William R. Schief, who is an associate professor of immunology and member of the IAVI Neutralizing Antibody Center at TSRI.

 





The researchers demonstrated their new technique by engineering a compound that has promise to initiate an otherwise rare immune response against many types of HIV. Here, the germline-targeting immunogen eOD-GT6 (red) is shown bound to its target, the germline VRC01 antibody (magenta and yellow). (Credit: Photo courtesy of The Scripps Research Institute.) 

 

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Scientists map protein that creates antibiotic resistance

Scientists map protein that creates antibiotic resistance | Power of Protein Crystal Structures | Scoop.it
Molecule changes shape to help organisms kick drugs out of cells. 

 

Japanese researchers have determined the detailed molecular structure of a protein that rids cells of toxins, but can also reduce the effectiveness of some antibiotics and cancer drugs by kicking them out of the cells they are targeting.

The scientists have also identified a molecule that can thwart the activity of the protein, one of a class known as multidrug and toxic compound extrusion transporters (MATEs) that are found in cell membranes. The discovery suggests new approaches to combat antibiotic resistance and boost the power of cancer therapies, the team reports today in Nature1.

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Synchrotron yields 'safer' vaccine

Synchrotron yields 'safer' vaccine | Power of Protein Crystal Structures | Scoop.it
British scientists develop a new way to create an entirely synthetic vaccine which does not rely on using live infectious virus, meaning it is much safer.

 

Producing vaccines against viral threats is a potentially hazardous business and that's why manufacturers have to operate strict controls to ensure that no pathogens escape.

British scientists have developed a new method to create an entirely synthetic vaccine which doesn't rely on using live infectious virus, meaning it is much safer.

What's more the prototype vaccine they have created, for the animal disease foot-and-mouth, has been engineered to make it more stable.

That means it can be kept out of the fridge for many hours before returning to the cold chain - overcoming one of the major hurdles in administering vaccines in the developing world.

The research, published in the journal PLOS pathogens, was a collaboration between scientists at Oxford and Reading Universities, the Pirbright Institute, and the UK's national synchrotron facility, the Diamond Light Source near Oxford.

                      Computer image of foot-and-mouth virus  
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Additional info...

http://www.guardian.co.uk/science/video/2013/mar/28/foot-and-mouth-vaccine-developed-video

 

http://www.guardian.co.uk/science/occams-corner/2013/mar/30/1

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New methodology for the analysis of proteins

New methodology for the analysis of proteins | Power of Protein Crystal Structures | Scoop.it
A study led by the professor of Biochemistry and Molecular Biology from the Faculty of Chemistry of the UB Modesto Orozco, and by Xavier Salvatella, from the Department of Biochemistry, both ICREA scientists at the Institute for Research in..

 

More information: Candotti, M. et al. Towards an atomistic description of the urea-denatured state of proteins. Proceedings of the National Academy of Sciences (PNAS), (early edition) 25th March 2013. DOI: 10.1073/pnas.1216589110

Read more at: http://phys.org/news/2013-04-methodology-analysis-proteins.html#jCp

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Insights from the sea: Structural biology of marine polyketide synthases

Insights from the sea: Structural biology of marine polyketide synthases | Power of Protein Crystal Structures | Scoop.it

The world's oceans are a rich source of natural products with extremely interesting chemistry.

Biosynthetic pathways have been worked out for a few, and the story is being enriched with crystal structures of interesting pathway enzymes. By far, the greatest number of structural insights from marine biosynthetic pathways has originated with studies of curacin A, a poster child for interesting marine chemistry with its cyclopropane and thiazoline rings, internal cis double bond, and terminal alkene. Using the curacin A pathway as a model, structural details are now available for a novel loading enzyme with remarkable dual decarboxylase and acetyltransferase activities, an Fe2+/α-ketoglutarate-dependent halogenase that dictates substrate binding order through conformational changes, a decarboxylase that establishes regiochemistry for cyclopropane formation, and a thioesterase with specificity for β-sulfated substrates that lead to terminal alkene offloading. The four curacin A pathway dehydratases reveal an intrinsic flexibility that may accommodate bulky or stiff polyketide intermediates. In the salinosporamide A pathway, active site volume determines the halide specificity of a halogenase that catalyzes for the synthesis of a halogenated building block. Structures of a number of putative polyketide cyclases may help in understanding reaction mechanisms and substrate specificities although their substrates are presently unknown.


Via NatProdChem
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First-ever determination of protein structure with X-ray laser

First-ever determination of protein structure with X-ray laser | Power of Protein Crystal Structures | Scoop.it
An international team of researchers, have, for the first time, used an ultra-intense X-ray laser to determine the previously unknown atomic-scale structure of a protein.
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Brighton researchers' discovery may prolong cancer patients’ lives

Cancer patients around the world could have their lives extended thanks to a discovery made by University of Sussex scientists

 

Cancer patients around the world could have their lives extended thanks to a discovery made by Sussex scientists.

 

Researchers at the University of Sussex, who have been working with the Institute of Cancer Research, have found that a cutting-edge cancer drug may be able to keep patients alive for longer than they live now.

The discovery by the researchers, who looked at exactly how the drugs attack tumours, has been hailed as “unexpected and exciting”.

The drugs, known as kinase inhibitors, are a new type of treatment, with 25 currently in use on a variety of cancers. 

Another 400 are under development.

Around 5,000 to 10,000 patients receive the drugs in the UK each year, with that number set to grow as more of the drugs are approved for use.

Kinase inhibitors work across types of breast, skin, lung and kidney cancer, but often only extend life by around three to six months.

Researchers believe they can unlock the true potential of the drugs by changing the way they are used – after uncovering a hidden way that they work.

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Protein misfolding and disease | The Scicurious Brain, Scientific American Blog Network

Protein misfolding and disease | The Scicurious Brain, Scientific American Blog Network | Power of Protein Crystal Structures | Scoop.it

Lots of people set themselves goals – like things to do by the time you’re 30. Maybe it’s to find your dream job, meet the love of your life, or travel the world! For sufferers of Cystic Fibrosis, it’s living to see your 30th birthday. Even with all of the advances in medicine and technology, the average life expectancy of someone with Cystic Fibrosis is 33 years. Cystic Fibrosis is an inherited disease that mostly affects the lungs, but also the pancreas, liver and intestines. The body fluids we need – like the mucus in our lungs and intestines – are much thicker than normal, making it extremely difficult to breathe and digest food. Constant physiotherapy, breathing exercises, diet supplements and antibiotics are needed just to get on with daily life. And all of this suffering is caused by one tiny change in our DNA, which then messes up how one single protein folds into the right shape. It’s otherwise known as a protein misfolding disease.

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Researchers uncover structure of new protein implicated in diabetes

Researchers uncover structure of new protein implicated in diabetes | Power of Protein Crystal Structures | Scoop.it
(Phys.org) —Scientists at the U.S. Department of Energy's Argonne National Laboratory, in collaboration with researchers from the Sanford-Burnham Medical Research Institute in Orlando, Fla., recently determined and analyzed the three-dimensional..

 

Scientists at the U.S. Department of Energy's Argonne National Laboratory, in collaboration with researchers from the Sanford-Burnham Medical Research Institute in Orlando, Fla., recently determined and analyzed the three-dimensional structure of a protein found in the nuclei of liver and pancreatic cells. The protein, called hepatocyte nuclear factor 4α (HNF-4α), plays a critical role by binding to specific DNA sequences and regulating the production of a number of key proteins for normal cellular processes. Some of its mutations have been linked to maturity-onset diabetes, kidney failure and metabolic syndrome.


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Mass spectrometry of intact protein complexes : Nature Methods : Nature Publishing Group

Mass spectrometry of intact protein complexes : Nature Methods : Nature Publishing Group | Power of Protein Crystal Structures | Scoop.it
Mass spectrometry technology to detect and characterize large, intact protein complexes is becoming more accessible.
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Taking the crystals out of X-ray crystallography

Taking the crystals out of X-ray crystallography | Power of Protein Crystal Structures | Scoop.it
Tiny molecular sponges can hold small molecules in position for imaging.

 

The technique that revealed DNA's double helix and the shapes of thousands of other molecules is getting an upgrade.

A method described in Nature this week1 makes X-ray crystallography of small molecules simpler, faster and more sensitive, largely doing away with the laborious task of coaxing molecules to form crystals. Instead, porous scaffolding holds molecules in the orderly arrangement needed to discern their structure with X-rays.

"You could call it crystal-free crystallography," says Jon Clardy, a biological chemist at Harvard Medical School in Boston, Massachusetts, who was not involved in the work but wrote a commentary accompanying the paper2.

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SX-Ray Crystallography: 100 Years at the Intersection of Physics, Chemistry, and Biology

SX-Ray Crystallography: 100 Years at the Intersection of Physics, Chemistry, and Biology | Power of Protein Crystal Structures | Scoop.it
We're having this month's Scicurious Guest Writer a little early, to make sure he gets some exposure and to avoid the holiday rush! Please welcome Satchal ...

 

In the summer of 1912, a young man and his father worked feverishly to interpret the results of a German physicist. The physicist, future Nobel Laureate Max von Laue, had recently observed the behavior of X-rays when exposed on a crystal, and was struggling to describe the interference of X-ray waves that resulted. Though the solution eluded him, the discovery, which soon would give birth to one of the most important techniques in science, came at an exciting time. The 19th and early 20th century marked an unusually active and competitive era in science; prior to this, it was believed that much of scientific theory had been conclusively worked out, particularly in the physical sciences. However, in the early 1900s, a number of brilliant researchers made startling breakthroughs that showed we were just scratching the surface of our scientific knowledge. It was in this vigorous environment that the young man, William Lawrence Bragg, then a graduate student on his summer break, and his father, William Henry Bragg, a mathematician and physicist, scrambled to understand a series of observations made by von Laue.

Upon his return to graduate school at Cambridge, where he was studying mathematics, the younger Bragg had made his breakthrough, and his doctoral adviser, the Nobel Laureate J.J. Thomson, presented his results on 11 November 1912 to the Cambridge Philosophical Society. Bragg had deduced what von Laue had struggled with: how individual spots on an X-ray diffraction pattern related to the atomic structure of the crystal that scattered them. Bragg’s formulation, now known as Bragg’s Law, successfully identified these positions. This meant, in essence, that, by crystallizing molecules and exposing the crystals to X-rays, the structures of individual molecules could be determined. The result was a seminal moment in the history of science, the birth of what remains today the most accurate technique to determine molecular structures: X-ray Crystallography. For his part in discovering X-ray diffraction from crystals, von Laue won the Nobel Prize in Physics in 1914, while both W.H. and W.L Bragg won the Nobel Prize in Physics in 1915 for the formulation. Since then hundreds of thousands of molecular structures have been determined via X-ray crystallography, with important consequences for physics, chemistry, and biology. The centenary gives us an opportunity to look back on not just Bragg but on the many great researchers who followed.

 

 

 

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