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  • richardmitnick 5:57 am on March 10, 2015 Permalink | Reply
    Tags: , , , Protein Studies,   

    From Scripps: “TSRI Scientists Reveal Structural Secrets of Nature’s Little Locomotive” 

    Scripps

    Scripps Research Institute

    March 9, 2015
    No Writer Credit

    Findings Could Help Shed Light on Alzheimer’s, Parkinson’s, ALS and Other Diseases

    A team led by scientists at The Scripps Research Institute (TSRI) has determined the basic structural organization of a molecular motor that hauls cargoes and performs other critical functions within cells.

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    The new research provides the first picture of a molecular motor called the “dynein-dynactin complex,” which is critical for cell division and cargo transport. (Image courtesy of the Lander lab, The Scripps Research Institute.)

    Biologists have long wanted to know how this molecular motor—called the “dynein-dynactin complex”—works. But the complex’s large size, myriad subunits and high flexibility have until now restricted structural studies to small pieces of the whole.

    In the new research, however, TSRI biologist Gabriel C. Lander and his laboratory, in collaboration with Trina A. Schroer and her group at Johns Hopkins University, created a picture of the whole dynein-dynactin structure.

    “This work gives us critical insights into the regulation of the dynein motor and establishes a structural framework for understanding why defects in this system have been linked to diseases such as Huntington’s, Parkinson’s, and Alzheimer’s,” said Lander.

    The findings are reported in a Nature Structural & Molecular Biology advance online publication on March 9, 2015.

    Unprecedented Detail

    The proteins dynein and dynactin normally work together on microtubules for cellular activities such as cell division and intracellular transport of critical cargo such as mitochondria and mRNA. The complex also plays a key role in neuronal development and repair, and problems with the dynein-dynactin motor system have been found in brain diseases including Alzheimer’s, Parkinson’s and Huntington’s diseases, and amyotrophic lateral sclerosis (ALS). In addition, some viruses (including herpes, rabies and HIV) appear to hijack the dynein-dynactin transport system to get deep inside cells.

    “Understanding how dynein and dynactin interact and work, and how they actually look, is definitely going to have medical relevance,” said Research Associate Saikat Chowdhury, a member of the Lander lab who was first author of the study.

    To study the dynein-dynactin complex, Schroer’s laboratory first produced individual dynein and dynactin proteins, which are themselves complicated, with multiple subunits, but have been so highly conserved by evolution that they are found in almost identical form in organisms from yeast to mammals.

    Chowdhury and Lander then used electron microscopy (EM) and cutting-edge image-processing techniques to develop two-dimensional “snapshots” of dynein’s and dynactin’s basic structures. These structural data contained unprecedented detail and revealed subunits never observed before.

    Chowdhury and Lander next developed a novel strategy to purify and image dynein and dynactin in complex together on a microtubule—a railway-like structure, ubiquitous in cells, along which dynein-dynactin moves its cargoes.

    “This is the first snapshot of how the whole dynein-dynactin complex looks and how it is oriented on the microtubule,” Chowdhury said.

    Pushing the Limits

    The structural data clarify how dynein and dynactin fit together on a microtubule, how they recruit cargoes and how they manage to move those cargoes consistently in a single direction.

    Lander and Chowdhury now hope to build on the findings by producing a higher-resolution, three-dimensional image of the dynein-dynactin-microtubule complex, using an EM-related technique called electron tomography.

    “The EM facility at TSRI is the best place in the world to push the limits of imaging complicated molecular machines like these,” said Lander.

    The other co-author of the paper, Structural organization of the dynein–dynactin complex bound to microtubules, (doi:10.1038/nsmb.2996) was Stephanie A. Ketcham of the Schroer laboratory.

    The research was supported by the Damon Runyon Cancer Research Foundation (DFS-#07-13), the Pew Scholars program, the Searle Scholars program and the National Institutes of Health (DP2 EB020402-01, GM44589).

    See the full article here.

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    The Scripps Research Institute (TSRI), one of the world’s largest, private, non-profit research organizations, stands at the forefront of basic biomedical science, a vital segment of medical research that seeks to comprehend the most fundamental processes of life. Over the last decades, the institute has established a lengthy track record of major contributions to the betterment of health and the human condition.

    The institute — which is located on campuses in La Jolla, California, and Jupiter, Florida — has become internationally recognized for its research into immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune diseases, cardiovascular diseases, virology, and synthetic vaccine development. Particularly significant is the institute’s study of the basic structure and design of biological molecules; in this arena TSRI is among a handful of the world’s leading centers.

    The institute’s educational programs are also first rate. TSRI’s Graduate Program is consistently ranked among the best in the nation in its fields of biology and chemistry.

     
  • richardmitnick 6:35 am on March 8, 2015 Permalink | Reply
    Tags: , , Protein Studies, Proteomics   

    From ETH: “Personalising medicine with proteins” 

    ETH Zurich bloc

    ETH Zurich

    06.03.2015
    Fabio Bergamin

    Ruedi Aebersold, Professor of Systems Biology, is one of the world’s leading researchers in proteomics. In the last few years, he has developed the proteomics method together with a team of international researchers to such an extent that doctors and clinical researchers can now use this technique as a tool. In an interview with ETH News, the professor at ETH Zurich and the University of Zurich explains how information from proteins can advance what is commonly known as personalised medicine.

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    “Proteins are the molecular players in cells, not genes,” says Ruedi Aebersold, professor at ETH and the University of Zurich. (Photo: Fabio Bergamin / ETH Zurich)

    ETH News: In future, medical researchers would like to better understand the individual differences among patients and the various manifestations of a disease in order to provide customised therapies. Until now, they have used mainly genomic differences; that is, mutations in the genetic material or DNA. Professor Aebersold, you are now going one step further and would like to entrench personalised medicine at the level of proteins. Why?

    Ruedi Aebersold: The molecular players that are the immediate cause of a disease in a body or a cell, are mostly proteins. Pathologists have long been able to measure specific proteins in tissue samples when they diagnose diseases; for example, a type of cancer. They can make these proteins visible with antibodies through the use of widely accepted, conventional methods. However, this identifies only a handful of proteins at a time. In recent years, we have developed a proteomics method by which we can precisely and simultaneously identify 2,000 proteins in a minute tissue sample.

    However, such methods to identify protein patterns are far more complex than existing genome analysis.
    It’s true that genetic mutations can now be identified quickly and relatively inexpensively. However, in cells the genetic information is processed; the proteins are at the end of the biological process chain and are more meaningful when describing a disease. By identifying thousands of proteins in tissue samples, we want to bridge the gap between genomics and diseases. Often a number of different genetic mutations lead to the same disease, or a disease is so complex with so many genetic puzzle pieces, of which we know very little about. Our proteomics method gives pathologists a modern tool with which they can classify diseased tissue far more precisely than before. We have developed proteomics to such an extent that we can deliver very precise and reproducible results in just one day.

    How do you achieve this?

    In order to identify proteins in a sample, we break them down into fragments called peptides. Using mass spectrometry, we can differentiate these peptides according to their mass and their ability to repel water.

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    We assume that there are 10 to 100 million different peptides that may arise from the different proteins in the human body, which is far too many to analyse in a short time. Many previous proteomics methods therefore used a trick: according to the Las Vegas principle, they randomly chose approximately every 1000th peptide and analysed it. But this method has the big disadvantage the data generated are poorly reproducible because the same peptides are not chosen each time the same sample is analyzed. We, on the other hand, reduce the size of the data in a different way: we divide the peptides according to their mass and ability to repel water into about 30,000 groups and analyse them within an hour. Chance plays no part in our method and our technique is therefore both reproducible and fast.

    Over the last two years, you have refined the method and recently used it on patient tissue samples for the first time. To what effect?

    In our latest study, we measured the biochemical state of small biopsies, specifically kidney cancer biopsies, that we received from co-autors of the study, physicians at the Kantonspital St. Gallen. We were able to reproduce the pathologist’s findings at the protein level extremely well. Our technique enables us to create digital protein fingerprints of the samples. The advantage is that these fingerprints can be re-analysed at a later date, which means that researchers can use our data years later if they are interested in the function of a specific protein.

    Why is the speed of the method important?

    Proteomics allow us to make new discoveries best when we statistically analyse data from a large number of people, called a cohort. If a method is fast, then it has the capacity to examine large cohorts.

    You lead a research group of systems biologists. How have doctors at hospitals responded to your new method?

    We have received positive feedback from clinical researchers, and we anticipate that pathologists will soon be using the method to make clinical decisions. Proteomics used to have a bad reputation among physicians because it was comparatively expensive and complex, and it suffered from the Las Vegas syndrome, the poor reproducibility. We have now corrected this and are therefore convinced that our method has huge potential in clinics. We submitted our latest research to a medical journal for publication rather than a biological journal in the hope that it will make physicians and medical researchers more aware of the benefits of our technique. We are also pleased that our method not only works on our equipment; researchers have already adapted it for other equipment.

    How will you further develop the method?

    We are constantly working on increasing the number of measurable proteins. We also want to develop the method in such a way that we can measure older tissue samples that have been conserved in formalin. We could then analyse stored samples from patients about whom the subsequent course of disease and the chosen therapy is known. This would allow us to detect correlations between protein patterns and the course of the disease.

    Everyone is talking about personalised medicine these days, and new national research programmes are taking place in the UK and the US. What is the situation in Switzerland in respect to research on personalised medicine?

    We are very well positioned in Switzerland to explore complex diseases with a systems approach, in part thanks to the well-developed systems biology research in this country. And a centre of personalised medicine already exists through “Hochschulmedizin Zürich”. But more incentives are needed in order for physicians, researchers and engineers to work together more effectively. Similar to Barack Obama’s recent announcement in the US, it would be desirable to have a national research programme for personalised medicine in Switzerland, too. Scientists prepared a widely supported proposal to incorporate a such into the next legislative programme in 2017. At the end of 2016, the national research programme for systems biology, Systemsx.ch, will also come to an end as scheduled, and a personalised health programme could be built on it.

    ________________________________________________________________________

    About Ruedi Aebersold

    Ruedi Aebersold (60) is a pioneer in proteomics and systems biology. The journal Analytical Scientist described him in 2013 as the world’s second-most influential scientist in analytical sciences. After graduating from the University of Basel, Aebersold held positions at the California Institute of Technology and the University of Washington. He has been a professor of Systems Biology at ETH Zurich and University of Zurich since 2000/01.

    ________________________________________________________________________

    See the full article here.

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    ETH Zurich campus

    ETH Zurich is one of the leading international universities for technology and the natural sciences. It is well known for its excellent education, ground-breaking fundamental research and for implementing its results directly into practice.

    Founded in 1855, ETH Zurich today has more than 18,500 students from over 110 countries, including 4,000 doctoral students. To researchers, it offers an inspiring working environment, to students, a comprehensive education.

    Twenty-one Nobel Laureates have studied, taught or conducted research at ETH Zurich, underlining the excellent reputation of the university.

     
  • richardmitnick 2:01 pm on February 3, 2015 Permalink | Reply
    Tags: , Protein Studies,   

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

    U Alberta bloc

    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.

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    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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    U Alberta Campus

    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.”

     
  • richardmitnick 8:02 am on January 24, 2015 Permalink | Reply
    Tags: , Protein Studies, UC Irvine   

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

    UC Irvine bloc

    UC Irvine

    January 23, 2015
    Janet Wilson, UC Irvine

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    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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    UC Irvine Campus

    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.

     
  • richardmitnick 6:39 am on January 20, 2015 Permalink | Reply
    Tags: , Hunger, Protein Studies,   

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

    UC Santa Cruz

    UC Santa Cruz

    January 19, 2015
    Tim Stephens

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    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.

     
  • richardmitnick 4:24 pm on December 4, 2014 Permalink | Reply
    Tags: , Protein Studies, ,   

    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.

    m
    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.

    SLAC LCLS
    SLAC LCLS Inside
    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.

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    SLAC Campus
    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.
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  • richardmitnick 4:31 pm on November 19, 2014 Permalink | Reply
    Tags: , , , Protein Studies   

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

    Berkeley Logo

    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.

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    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 Advanced Light Source
    LBL ALS interior
    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.

    gh
    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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  • richardmitnick 4:16 am on May 7, 2014 Permalink | Reply
    Tags: , , Protein Studies,   

    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.

    ps
    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.

    robotics
    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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  • richardmitnick 4:16 pm on March 21, 2014 Permalink | Reply
    Tags: , , , Protein Studies,   

    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
    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.

    sticky
    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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  • richardmitnick 3:47 pm on March 21, 2014 Permalink | Reply
    Tags: , , , Protein Studies,   

    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.

    protein
    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.”

    Brookhaven NSLS II Photo
    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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