From Research at Princeton: “New method identifies protein-protein interactions on basis of sequence alone (PNAS)”

Princeton University

September 23, 2016
Catherine Zandonella, Office of the Dean for Research

Researchers can now identify which proteins will interact just by looking at their sequences. Pictured are surface representations of a histidine kinase dimer (HK, top) and a response regulator (RR, bottom), two proteins that interact with each other to carry out cellular signaling functions. (Image based on work by Casino, et. al. credit: Bitbol et. al 2016/PNAS.)

Genomic sequencing has provided an enormous amount of new information, but researchers haven’t always been able to use that data to understand living systems.

Now a group of researchers has used mathematical analysis to figure out whether two proteins interact with each other, just by looking at their sequences and without having to train their computer model using any known examples. The research, which was published online today in the journal Proceedings of the National Academy of Sciences, is a significant step forward because protein-protein interactions underlie a multitude of biological processes, from how bacteria sense their surroundings to how enzymes turn our food into cellular energy.

“We hadn’t dreamed we’d be able to address this,” said Ned Wingreen, Princeton University‘s Howard A. Prior Professor in the Life Sciences, and a professor of molecular biology and the Lewis-Sigler Institute for Integrative Genomics, and a senior co-author of the study with Lucy Colwell of the University of Cambridge. “We can now figure out which protein families interact with which other protein families, just by looking at their sequences,” he said.

Although researchers have been able to use genomic analysis to obtain the sequences of amino acids that make up proteins, until now there has been no way to use those sequences to accurately predict protein-protein interactions. The main roadblock was that each cell can contain many similar copies of the same protein, called paralogs, and it wasn’t possible to predict which paralog from one protein family would interact with which paralog from another protein family. Instead, scientists have had to conduct extensive laboratory experiments involving sorting through protein paralogs one by one to see which ones stick.

In the current paper, the researchers use a mathematical procedure, or algorithm, to examine the possible interactions among paralogs and identify pairs of proteins that interact. The method was able to correctly predict 93% of the protein-protein paralog pairs that were present in a dataset of more than 20,000 known paired protein sequences, without being first provided any examples of correct pairs.

Interactions between proteins happen when two proteins come into physical contact and stick together via weak bonds. They may do this to form part of a larger piece of machinery used in cellular metabolism. Or two proteins might interact to pass a signal from the exterior of the cell to the DNA, to enable a bacterial organism to react to its environment.

When two proteins come together, some amino acids on one chain stick to the amino acids on the other chain. Each site on the chain contains one of 20 possible amino acids, yielding a very large number of possible amino-acid pairings. But not all such pairings are equally probable, because proteins that interact tend to evolve together over time, causing their sequences to be correlated.

The algorithm takes advantage of this correlation. It starts with two protein families, each with multiple paralogs in any given organism. The algorithm then pairs protein paralogs randomly within each organism and asks, do particular pairs of amino acids, one on each of the proteins, occur much more or less frequently than chance? Then using this information it asks, given an amino acid in a particular location on the first protein, which amino acids are especially favored at a particular location on the second protein, a technique known as direct coupling analysis. The algorithm in turn uses this information to calculate the strengths of interactions, or “interaction energies,” for all possible protein paralog pairs, and ranks them. It eliminates the unlikely pairings and then runs again using only the top most likely protein pairs.

The most challenging part of identifying protein-protein pairs arises from the fact that proteins fold and kink into complicated shapes that bring amino acids in proximity to others that are not close by in sequence, and that amino-acids may be correlated with each other via chains of interactions, not just when they are neighbors in 3D. The direct coupling analysis works surprisingly well at finding the true underlying couplings that occur between neighbors.

The work on the algorithm was initiated by Wingreen and Robert Dwyer, who earned his Ph.D. in the Department of Molecular Biology at Princeton in 2014, and was continued by first author Anne-Florence Bitbol, who was a postdoctoral researcher in the Lewis-Sigler Institute for Integrative Genomics and the Department of Physics at Princeton and is now a CNRS researcher at Universite Pierre et Marie Curie – Paris 6. Bitbol was advised by Wingreen and Colwell, an expert in this kind of analysis who joined the collaboration while a member at the Institute for Advanced Study in Princeton, NJ, and is now a lecturer in chemistry at the University of Cambridge.

The researchers thought that the algorithm would only work accurately if it first “learned” what makes a good protein-protein pair by studying ones discovered in experiments. This required that the researchers give the algorithm some known protein pairs, or “gold standards,” against which to compare new sequences. The team used two well-studied families of proteins, histidine kinases and response regulators, which interact as part of a signaling system in bacteria.

But known examples are often scarce, and there are tens of millions of undiscovered protein-protein interactions in cells. So the team decided to see if they could reduce the amount of training they gave the algorithm. They gradually lowered the number of known histidine kinase-response regulator pairs that they fed into the algorithm, and were surprised to find that the algorithm continued to work. Finally, they ran the algorithm without giving it any such training pairs, and it still predicted new pairs with 93 percent accuracy.

“The fact that we didn’t need a gold standard was a big surprise,” Wingreen said.

Upon further exploration, Wingreen and colleagues figured out that their algorithm’s good performance was due to the fact that true protein-protein interactions are relatively rare. There are many pairings that simply don’t work, and the algorithm quickly learned not to include them in future attempts. In other words, there is only a small number of distinctive ways that protein-protein interactions can happen, and a vast number of ways that they cannot happen. Moreover, the few successful pairings were found to repeat with little variation across many organisms. This it turns out, makes it relatively easy for the algorithm to reliably sort interactions from non-interactions.

Wingreen compared this observation – that correct pairs are more similar to one another than incorrect pairs are to each other – to the opening line of Leo Tolstoy’s Anna Karenina, which states, “All happy families are alike; each unhappy family is unhappy in its own way.”

The work was done using protein sequences from bacteria, and the researchers are now extending the technique to other organisms.

The approach has the potential to enhance the systematic study of biology, Wingreen said. “We know that living organisms are based on networks of interacting proteins,” he said. “Finally we can begin to use sequence data to explore these networks.”

The research was supported in part by the National Institutes of Health (Grant R01-GM082938) and the National Science Foundation (Grant PHY–1305525).

See the full article here .

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About Princeton: Overview

Princeton University is a vibrant community of scholarship and learning that stands in the nation’s service and in the service of all nations. Chartered in 1746, Princeton is the fourth-oldest college in the United States. Princeton is an independent, coeducational, nondenominational institution that provides undergraduate and graduate instruction in the humanities, social sciences, natural sciences and engineering.

As a world-renowned research university, Princeton seeks to achieve the highest levels of distinction in the discovery and transmission of knowledge and understanding. At the same time, Princeton is distinctive among research universities in its commitment to undergraduate teaching.

Today, more than 1,100 faculty members instruct approximately 5,200 undergraduate students and 2,600 graduate students. The University’s generous financial aid program ensures that talented students from all economic backgrounds can afford a Princeton education.

From ESRF: “#weekendusers Express experiments for better health”

The European Synchrotron

19-09-2016

Synchrotron sources are crucial for structural biology and research performed at the ESRF produces 40% of the protein structures submitted in Europe. This weekend, scientists from Barcelona spent 24 hours trying to unveil the structures of medically-relevant proteins.

They drove more than 1000km for 24h of experiments and 130 samples. The aim: to get insight into the structures of proteins that play a role in health, such as mycoplasma, viral and membrane transport proteins. “It is very intense”, explains David Aparicio, from the Institut de Biologia Molecular de Barcelona (Spain), “but we are now coming once a month for 24h each time, so we are used to this rythm”, he adds. He is one of the four scientists on ID23-1 this weekend. Two of them, Victor Ruiz and Diego Ferrero, also come from the IBMB, but from different research groups, whilst the last one, Ekaitz Errasti works in the Institute for Research in Biomedicine (IRB) in the same city.

Checking that the experiment is going smoothly in the small hours of Sunday. No image credit.

The researchers came to the ESRF in the framework of the so-called Block Allocation Group from Barcelona. Crystallographers from large, well-established groups requiring a significant amount of beamtime often present their proposals in groups. BAG Barcelona includes groups that belong to the Institute for Molecular Biology of Barcelona (IBMB) from the Spanish Reseach Council (CSIC), the Institute of Biomedical Research (IRB) and the Institut de Recerca – Hospital de la Santa Creu i Sant Pau (HSCSP).

Maria Solà, coordinator of the BAG Barcelona, explains that “the success of our projects is absolutely dependent on obtaining data using synchrotron radiation”, due to the size and characteristics of the samples. “The tunable and microfocus beamline of ID23 fulfills excellently all our requirements”, she adds, “so we are hoping for good data”.

See the full article here .

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The ESRF – the European Synchrotron Radiation Facility – is the most intense source of synchrotron-generated light, producing X-rays 100 billion times brighter than the X-rays used in hospitals. These X-rays, endowed with exceptional properties, are produced at the ESRF by the high energy electrons that race around the storage ring, a circular tunnel measuring 844 metres in circumference. Each year, the demand to use these X-ray beams increases and thousands of scientists from around the world come to Grenoble, to access the 43 highly specialised experimental stations, called “beamlines”, each equipped with state-of-the-art instrumentation, operating 24 hours a day, seven days a week.

Thanks to the brilliance and quality of its X-rays, the ESRF functions like a “super-microscope” which “films” the position and motion of atoms in condensed and living matter, and reveals the structure of matter in all its beauty and complexity. It provides unrivalled opportunities for scientists in the exploration of materials and living matter in a very wide variety of fields: chemistry, material physics, archaeology and cultural heritage, structural biology and medical applications, environmental sciences, information science and nanotechnologies.

Following on from 20 years of success and excellence, the ESRF has embarked upon an ambitious and innovative modernisation project, the Upgrade Programme, implemented in two phases: Phase I (2009-2015) and the ESRF-EBS (Extremely Brilliant Source) (2015-2022) programmes. With an investment of 330 million euros, the Upgrade Programme is paving the way to a new generation of synchrotron storage rings, that will produce more intense, coherent and stable X-ray beams. By constructing a new synchrotron, deeply rooted in the existing infrastructure, the ESRF will lead the way in pushing back the boundaries of scientific exploration of matter, and contribute to answering the great technological, economic, societal and environmental challenges confronting our society.

From ANL: “Two protein studies discover molecular secrets to recycling carbon and healing cells”

News from Argonne National Laboratory

September 9, 2016
Kate Thackrey

Researchers at Argonne modeled the HcaR protein complex, above, a sort of molecular policeman that controls when to activate genes that code for enzymes used by Acinetobacter bacteria to break down compounds for food. Understanding these processes can help scientists develop ideas for converting more carbon in soil. (Image courtesy Kim et al./Journal of Biological Chemistry.)

Researchers at the U.S. Department of Energy’s (DOE’s) Argonne National Laboratory have mapped out two very different types of protein. One helps soil bacteria digest carbon compounds; the other protects cells from the effects of harmful molecules.

In order to uncover the structure of these proteins, researchers used a technique called protein crystallography. Like a mosquito trapped in amber, compounds that are crystallized are placed in array in identical positions and ordered so that scientists can target them with X-ray beams and work backwards from the scattering patterns produced to recreate their three-dimensional structures atom by atom.

Bacterial breakdown

In the first study, a group of researchers from the Structural Biology Center, which is funded by DOE’s Office of Science, mapped out a protein responsible for breaking down organic compounds in soil bacteria, an important process for recycling carbon in the ecosystem.

The bacteria used, called Acinetobacter, is located mostly in soil and water habitats, where it helps to change aromatic compounds (named for their ring shape) into forms that can be used as food.

One of the sources of aromatic compounds found in soil is lignin, a tough polymer that is an essential part of all plants and that’s hard for many organisms to digest.

“But Acinetobacter can utilize these aromatic compounds as their sole source of carbon,” said Andrzej Joachimiak, who co-authored both studies and is the director of the Structural Biology Center and the Midwest Center for Structural Genomics at Argonne.

In order for Acinetobacter to break down the aromatic compounds, it needs to produce catabolic enzymes, molecular machines built from an organism’s DNA that break down molecules into smaller parts that can be digested.

Whether or not membrane transporters and catabolic enzymes are produced falls to the HcaR regulator, a sort of molecular policeman that controls when the genes that code for these enzymes can be activated.

Joachimiak and his colleagues found that the regulator works in a cycle, activating genes when aromatic compounds are present and shutting genes down when the compounds are used up.

“By nature it is very efficient,” Joachimiak said. “If you don’t have aromatic compounds inside a cell, the operon is shut down.”

The research team didn’t stop at mapping out the regulator itself; to discover how the cycle worked, they crystalized the HcaR regulator during interactions with its two major inputs: the aromatic compounds and DNA.

The group found that when aromatic compounds are not present in the cell, two wings found on either side of the HcaR regulator wrap around the DNA. This action is mirrored on both sides of the regulator, covering the DNA regulatory site and preventing genes from being activated.

“This is something that has never been seen before,” Joachimiak said.

When the aromatic compounds are present, however, they attach themselves to the HcaR regulator, making it so stiff that it can no longer grapple with the DNA.

Joachimiak said that this knowledge could help outside of the lab, with applications such as a sensor for harmful pesticides and as a template for converting more carbon in soil.

“If we can train bacteria to better degrade lignin and other polymers produced by plants during photosynthesis, more natural carbon sources can be utilized for example for production of biofuels and bioproducts,” Joachimiak said.

The paper was published earlier this year by the Journal of Biological Chemistry under the title How Aromatic Compounds Block DNA Binding of HcaR Catabolite Regulator. It was supported by the National Institutes of Health and the U.S. Department of Energy (Office of Biological and Environmental Research).

Protective proteins

A second paper focuses on a family of proteins identified as DUF89, which stands for “domain unknown function.” This family is conserved across all three branches of the phylogenetic tree, which means that it is likely essential to many life forms.

DUF89 has been identified as a type of enzyme called a phosphatase, which strips molecules of their phosphate groups. The paper’s authors hypothesized that DUF89 proteins use this ability to save useful proteins in a cell from rogue molecules which could alter their structure, making them useless or destructive.

The study found that DUF89 proteins use a metal ion, probably manganese, to lure in potentially harmful molecules and a water molecule to break off their phosphate group.

DUF89 proteins could have an important role in breaking down a specific type of disruptive molecule: sugar. When the concentration of sugar in blood reaches high levels, simple sugars can have unwanted side reactions with proteins and DNA through a process called glycation.

“We always have to deal with these side reactions that happen in our cells, and when we get older, we have an accumulation of these errors in our cells,” Joachimiak said.

Joachimiak said that this research could help scientists develop DUF89 treatments from non-human sources as a way to combat glycation in the bloodstream.

The paper was published on the Nature Chemical Biology website on June 20 under the title A family of metal-dependent phosphatases implicated in metabolite damage-control. Other authors on the paper were from the University of Florida, the University of Toronto, the University of California-Davis and Brookhaven National Laboratory. It was supported by the National Science Foundation, Genome Canada, the Ontario Genomics Institution, the Ontario Research Fund, the Natural Sciences and Engineering Research Council of Canada, the National Institutes of Health, the C.V. Griffin Sr. Foundation and the U.S. Department of Energy (Office of Basic Energy Sciences and Office of Biological and Environmental Research).

Both studies used X-rays from the Advanced Photon Source, a DOE Office of Science User Facility, using beamlines 19-ID and 19-BM.

Both also stem from the goal of the Midwest Center for Structural Genomics, which is to discover the structure and function of proteins potentially important to biomedicine.

Joachimiak said that despite the new findings from these studies, when it comes to understanding what proteins do, we still have a long way to go.

“When we sequence genomes, we can predict proteins, but when we predict those sequences we can only say something about function for about half of them,” Joachimiak said.

See the full article here .

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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. For more visit http://www.anl.gov.

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. To learn more about the Office of Science X-ray user facilities, visit http://science.energy.gov/user-facilities/basic-energy-sciences/.

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

From ICL: “A new way to create synthetic proteins could lead to more flexible designs”

Imperial College London

Structure of a designed protein

Building up proteins from scratch, rather than piecing together fragments of existing proteins, could make designing new nanomaterials easier.

Proteins perform a myriad of functions essential for life. They also make up important and useful biological materials, for example spider silk, which is exceptionally strong but still flexible.

The ability to design completely new proteins would help scientists create nanomaterials that, like spider silk, have a specific microstructure that confers useful properties.

Until now, new proteins have usually been designed by piecing together fragments of existing proteins in order to simplify the design process.

Now, a team led by researchers from Imperial College London has used a synthetic repeating protein scaffold as a base and shown that it is possible to add individual computationally designed modules, which can be chosen for their ability to perform a specific function. This gives biological engineers the possibility of designing new molecules from scratch.

The base scaffold is a new artificial repeating helix to which functional modules can be added. The team designed the structure on a computer, created it using synthetic genes, and then used a technique called X-ray crystallography to confirm they had built what they set out to.

Building complexity

Study leader Dr James Murray from the Department of Life Sciences at Imperial said: “Our system would allow designers to create proteins with atoms in specific places and build up complexity module by module, rather than designing the protein all at once.”

Dr James MacDonald from Imperial’s Centre for Synthetic Biology and Innovation added: “We have developed a new method for computationally designing brand new proteins that is potentially more flexible than taking sections from known proteins.”

The team’s first experimental results, published today in Proceedings of the National Academy of Sciences, added just one module to a helical scaffold as a proof that the system could work. Next, they want to add more loops to build up new functionality, and then test whether the synthetic proteins perform as expected.

Professor Paul Freemont, co-founder and co-director of the Centre for Synthetic Biology and Innovation at Imperial, said: “Being able to construct proteins at the atomic level has a lot of potential and exciting applications, including synthetic enzymes and new nanomaterials.

These could include improved nanowire batteries, where viruses are programmed to produce thin wires that increase the surface area and performance of batteries.”

See the full article here .

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Imperial College London is a science-based university with an international reputation for excellence in teaching and research. Consistently rated amongst the world’s best universities, Imperial is committed to developing the next generation of researchers, scientists and academics through collaboration across disciplines. Located in the heart of London, Imperial is a multidisciplinary space for education, research, translation and commercialisation, harnessing science and innovation to tackle global challenges.

From Caltech: Women in STEM – “Multitasking Protein Keeps Immune System Healthy” Beth Stadtmueller

Caltech

09/01/2016
Lori Dajose

Simplified diagram of pIgR binding to an antibody. A) pIgR and an antibody. B) Recognition binding. pIgR chemically recognizes an antibody. C) Conformational change. The pIgR protein opens up. D) The bound state of pIgR and an antibody. Credit: B. Stadtmueller

Schematic summary highlighting the differences in pIgR structure among fish, birds and humans.
Credit: B. Stadtmueller

The polymeric immunoglobulin receptor, or pIgR, is a multitasking protein produced in the lining of mucosal surfaces, such as the intestines. It plays a pivotal role in the body’s immune functions by sequestering bacteria and by assisting antibodies—large proteins that can identify and neutralize specific bacteria and viruses. Now, scientists at Caltech have determined the three-dimensional structure of pIgR, providing important insights into how the protein keeps the immune system running smoothly.

Beth Stadtmueller, a postdoctoral scholar in the laboratory of Centennial Professor of Biology Pamela Björkman, is the first author on two recent papers describing the findings.

Beth Stadtmueller

“Proteins such as pIgR are folded into complicated shapes,” says Stadtmueller. “Having a complete model of a protein is analogous to an architectural model of a building showing scaled dimensions of walls, the locations of windows and doors, angles of the roof, and so on. Understanding the structure of this protein provides information on how it carries out normal functions while also providing a basis to rationally engineer modified proteins with enhanced functions, which could be used as therapeutics.”

The pIgR protein is best known for attaching to antibodies and ferrying them from the bloodstream to the interior of the intestines, where the antibodies can neutralize pathogens. In mammals such as humans, the group discovered that pIgR looks like five round beads—biologists call these regions “domains”—that are connected to form a tightly closed, triangle-shaped loop. The group also showed that upon encountering an antibody, the pIgR molecule opens up—like changing from a fist to an open hand—to enclose around the antibody and to transport it into the intestines.

While pIgR is crucial for helping antibodies to function, the protein also has disease-fighting abilities of its own. For example, some molecules of pIgR are released into the intestines where they alone engage bacteria, such as pneumonia-causing Streptococcus pneumoniae.

The group also studied the structures of pIgR from fish and birds, to see how the protein has changed as vertebrates evolved. In fish, pIgR has only two domains and forms a straight line. In birds, an evolutionary intermediary between fish and humans, the protein has four domains. The group was surprised to find that the shape of the bird pIgR is not fixed in a closed loop or a straight line—it can change freely between closed and open configurations, and can grasp antibodies much like the human protein.

“The human pIgR is like a door that has to be unlocked to open, whereas the bird pIgR is constantly opening and closing like a revolving door,” Stadtmueller says. “These are very different structures, which are likely to support functions unique to each protein.”

“The immune system has changed considerably as vertebrates have evolved,” she adds. “Studying pIgR in a spectrum of vertebrates illustrates how the protein architecture has changed to support species-specific defense systems. It helps us to understand why certain immune system functions have evolved and provides a foundation to test their contributions to specific states of health and disease.”

The three-dimensional structure of human pIgR is described in a March 2016 paper published in the journal eLife, titled The structure and dynamics of secretory component and its interactions with polymeric immunoglobulins. A follow-up study, titled Biophysical and biochemical characterization of avian secretory component provides structural insights into the evolution of the polymeric Ig receptor, describing the structure of avian pIgR, was published in the Journal of Immunology on August 15, 2016. The work was done in collaboration with the Hubbell laboratory at UCLA and supported by grants from the National Institute of Allergy and Infectious Diseases, the Cancer Research Irving Postdoctoral Fellowship, the Jules Stein Professorship Endowment, and the National Institutes of Health.

See the full article here .

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The California Institute of Technology (commonly referred to as Caltech) is a private research university located in Pasadena, California, United States. Caltech has six academic divisions with strong emphases on science and engineering. Its 124-acre (50 ha) primary campus is located approximately 11 mi (18 km) northeast of downtown Los Angeles. “The mission of the California Institute of Technology is to expand human knowledge and benefit society through research integrated with education. We investigate the most challenging, fundamental problems in science and technology in a singularly collegial, interdisciplinary atmosphere, while educating outstanding students to become creative members of society.”

From UCLA: “UCLA researchers develop method to speed up detection of infectious diseases, cancer”

UCLA

August 26, 2016
Matthew Chin

UCLA researchers were able to use a molecular chain reaction to detect the presence of proteins in blood and plasma in a way that is faster and simpler.

A team of UCLA researchers has found a way to speed and simplify the detection of proteins in blood and plasma opening up the potential for diagnosing the early presence of infectious diseases or cancer during a doctor’s office visit. The new test takes about 10 minutes as opposed to two to four hours for current state-of-the-art tests.

The new approach overcame several key challenges in detecting proteins that are biomarkers of disease. First, these proteins are often at low abundance in body fluids and accurately identifying them requires amplification processes. The current approach uses enzymes to amplify the signal from proteins. However, enzymes can break down if they are not stored at proper temperatures. Also, to avoid a false positive, excess enzymes need to be washed away. This increases the complexity and cost of the test.

The study, which included researchers from the Henry Samueli School of Engineering and Applied Science, the California NanoSystems Institute, and the David Geffen School of Medicine, was published online in the journal ACS Nano.

The researchers included lead author Donghyuk Kim, a UCLA post-doctoral researcher in bioengineering and Dino Di Carlo, professor of bioengineering. They collaborated with Aydogan Ozcan, Chancellor’s Professor of Electrical Engineering and Bioengineering and Omai Garner, assistant professor of pathology and medicine at the David Geffen School of Medicine at UCLA.

The UCLA team devised an approach to amplify a protein signal without any enzymes, thus eliminating the need for a complex system to wash away excess enzymes, and that would work only in the presence of the target protein. This new approach made use of a molecular chain reaction that was strongly triggered only in the presence of a target protein.

The molecular chain reaction is driven by a cycle of DNA binding events. The process begins with a DNA key divided into two parts. If the target protein is present, the two parts bind together to form a DNA complex. The formation of the DNA complex generates DNA signaling molecules, which in turn generates the same DNA complex, leading to more signaling molecules, thus propagating repeated cycles.

“By cutting the DNA ‘key’ into two parts, we found that each part could not catalyze or ‘open’ the reaction separately, but only when a protein acted as glue — essentially bridging the parts together, does the DNA key became functional again,” said Kim, a member of Di Carlo’s laboratory.

The UCLA team’s findings build on previous work that employed this enzyme-free mechanism of nucleic acid amplification to detect DNA.

“Unlike previous approaches to achieve an amplified readout of proteins, such as the proximity ligation assay, this approach does not require multiple enzymes, longer polymerization-based enzymatic reactions, or temperature control to amplify signal,” Di Carlo said. “In fact the new assay operates at room temperature and achieves results in about 10 minutes.”

The team demonstrated the approach with two target proteins — streptavidin, widely used as a test protein for new diagnostic assays, and influenza nucleoprotein, which is a protein associated with the influenza virus.

In the long term the team aims to combine the technique with portable readers that could be particularly beneficial in clinics in resource-poor areas.

“Because the technique requires fewer steps than other assays, it can have a significant impact on distributed diagnostics and public health reporting, especially in combination with cost-effective portable and networked reader technology that our lab is developing,” Ozcan said.

The team demonstrated a synergistic handheld microplate reader suitable for protein diagnostic assays based on a cellphone’s optical and computational systems earlier this year.

Garner, who is also the associate director of the clinical microbiology lab at UCLA Health, emphasized the broad application of the technique. “Although demonstrated initially in detecting protein associated with flu, we envision the approach can be generalized to a range of protein biomarkers associated with infectious diseases and cancer,” said Garner. He noted it could be configured to detect diseases such as Zika or Ebola.

The researchers emphasized that additional work is required to adapt the assay to complex clinical samples that may have other interfering compounds, and further optimization of the reagents for the assay can enhance performance.

This interdisciplinary work was supported through a team science grant from the National Science Foundation Emerging Frontiers in Research and Innovation program.

See the full article here .

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For nearly 100 years, UCLA has been a pioneer, persevering through impossibility, turning the futile into the attainable.

We doubt the critics, reject the status quo and see opportunity in dissatisfaction. Our campus, faculty and students are driven by optimism. It is not naïve; it is essential. And it has fueled every accomplishment, allowing us to redefine what’s possible, time after time.

This can-do perspective has brought us 12 Nobel Prizes, 12 Rhodes Scholarships, more NCAA titles than any university and more Olympic medals than most nations. Our faculty and alumni helped create the Internet and pioneered reverse osmosis. And more than 100 companies have been created based on technology developed at UCLA.

From TUM: “A look at the molecular quality assurance within cells”

Techniche Universitat Munchen

25.08.2016
Technical University of Munich
Prof. Dr. Matthias J. Feige
matthias.feige@tum.de
+49-89-28910595
http://www.cell.ch.tum.de

A look at the molecular quality assurance within cells. (Illustration: Joshua Stokes, St. Jude Children’s Research Hospital)

Proteins fulfill vital functions in our body. They transport substances, combat pathogens, and function as catalysts. In order for these processes to function reliably, proteins must adopt a defined three-dimensional structure. Molecular “folding assistants”, called chaperones, aid and scrutinize these structuring processes. With participation from the Technical University of Munich (TUM), a team of researchers has now revealed how chaperones identify particularly harmful errors in this structuring process. The findings were published in the scientific journal “Molecular Cell”.

Chaperones are a kind of Technical Inspection Authority for cells. They are proteins that inspect other proteins for quality defects before they are allowed to leave the cell.

If a car does not pass its technical inspection, it implies that it has severe defects that could lead to serious accidents. If a protein folds into a faulty structure, this may lead to serious diseases. Examples of these are neurodegenerative disorders such as Alzheimer’s, but also metabolic diseases such as cystic fibrosis and diabetes.

Matthias Feige, professor for cellular protein biochemistry at the TUM, worked within a team headed by Linda Hendershot at St. Jude Children’s Research Hospital in Memphis/TN, USA, to investigate how chaperones identify structurally flawed proteins. In the study, the scientists focused on proteins which are produced in a part of the cell called the endoplasmic reticulum. “We are mainly interested in cellular protein folding”, explains Feige. “How the self-organization of proteins occurs at the molecular level – and how cells identify errors in this process – is a truly fascinating question.”

Defective proteins need to be eliminated by the cell

The endoplasmic reticulum consists of a network of hollow spaces and tubules. It is specialized in protein folding and the quality control for this process, and a third of all human proteins are produced here. Just like in any production process, errors may occur: Proteins form a folding core mostly made up of hydrophobic (water-repellent) amino acids, around which the rest of the protein is able to structure itself. However, if errors occur in the folding process, these hydrophobic areas may not be buried in the core, but instead be exposed on the surface of a protein where they may result in proteins clumping together. This can become hazardous to the cell or the entire organism.

Into the cell via a shuttle

Thus far, scientists knew that chaperones were able to identify general hydrophobic amino acid sequences if they remained exposed on protein surfaces. However, not all proteins which present such sequences should necessarily be degraded. That is because not all proteins with hydrophobic amino acid sequences on the surface are defective. How exactly the cell decides if a protein is so dangerous that it needs to be eliminated remained a mystery.

The researchers developed a new method which made it possible to observe the behavior of chaperones in the living biological system of the cell. To do this, they inserted precisely defined sequences of amino acids, which are the building blocks of proteins, into a shuttle system that transported them into the endoplasmic reticulum within the cell. Via this ingenious trick, they were able to observe, under biologically relevant conditions, which sequences the various chaperones recognized.

Two classes of chaperones

What they discovered was that there existed not only one, but two classes of chaperones in the endoplasmic reticulum, each of which identifies different types of hydrophobic amino acid sequences. Furthermore, the sequences identified by the chaperones of the second class, which are described in this journal article for the first time, form particularly dangerous clumps in the cell. Once they are identified, the proteins possessing them can be eliminated rapidly.

“This is an important piece in the puzzle of how molecular quality control functions”, says Feige. “Follow-up studies will now be required to see how the chaperones recognize their target sequences on a structural level.”

This research is also important for the biotechnological production of proteins, such as antibodies. In order to prevent these pharmaceutical products from being broken down by the body too quickly, biotechnologists can now ensure that the corresponding sequences do not appear on the surface of the proteins.

Publication: Julia Behnke, Melissa J. Mann, Fei-Lin Scruggs, Matthias J. Feige, Linda M. Hendershot, “Members of the Hsp70 Family Recognize Distinct Types of Sequences to Execute ER Quality Control“, Molecular Cell, published online August 18, 2016

See the full article here .

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Technische Universität München (TUM) is one of Europe’s top universities. It is committed to excellence in research and teaching, interdisciplinary education and the active promotion of promising young scientists. The university also forges strong links with companies and scientific institutions across the world. TUM was one of the first universities in Germany to be named a University of Excellence. Moreover, TUM regularly ranks among the best European universities in international rankings.

From Rosetta@home: “Designed Protein Containers Push Bioengineering Boundaries”

Rosetta@home

Rosetta@home has posted in their forum a new (July 21, 2016) article, Designed Protein Containers Push Bioengineering Boundaries
from U Washington’s Institute for Protein Design which I highly recommend for anyone interested in Protein Studies.

This forum article cites Designed Protein Containers Push Bioengineering Boundariess which goes on to cite Icosahedral protein nanocage – new paper and podcast published in Nature, and “Accurate design of megadalton-scale multi-component icosahedral protein complexes”, published in Science.

Of this second paper, they write, “In this paper, former Baker lab graduate student Jacob Bale, Ph.D. and collaborators describe the computational design and experimental characterization of ten two-component protein complexes that self-assemble into nanocages with atomic-level accuracy. These nanocages are the largest designed proteins to date with molecular weights of 1.8-2.8 megadaltons and diameters comparable to small viral capsids. The structures have been confirmed by X-ray crystallography (see figure). The advantage of a multi-component protein complex is the ability to control assembly by mixing individually prepared subunits. The authors show that in vitro mixing of the designed subunits occurs rapidly and enables controlled packaging of negatively charged GFP by introducing positive charges on the interior surfaces of the two copmonents.

The ability to design, with atomic-level precision, these large protein nanostructures that can encapsulate biologically relevant cargo and that can be genetically modified with various functionalities opens up exciting new opportunities for targeted drug delivery and vaccine design.”

Also referenced in the forum is an article in Science, Jul. 21, 2016 by Robert F. Service This protein designer aims to revolutionize medicines and materials, about Dr David Baker.

From this Science article, David Baker shows off models of some of the unnatural proteins his team has designed and made.© Rich Frishman

included also is this video from Science.

See the full article here.

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Rosetta@home needs your help to determine the 3-dimensional shapes of proteins in research that may ultimately lead to finding cures for some major human diseases. By running the Rosetta program on your computer while you don’t need it you will help us speed up and extend our research in ways we couldn’t possibly attempt without your help. You will also be helping our efforts at designing new proteins to fight diseases such as HIV, Malaria, Cancer, and Alzheimer’s (See our Disease Related Research for more information). Please join us in our efforts! Rosetta@home is not for profit.

About Rosetta

One of the major goals of Rosetta is to predict the shapes that proteins fold up into in nature. Proteins are linear polymer molecules made up of amino acid monomers and are often refered to as “chains.” Amino acids can be considered as the “links” in a protein “chain”. Here is a simple analogy. When considering a metal chain, it can have many different shapes depending on the forces exerted upon it. For example, if you pull its ends, the chain will extend to a straight line and if you drop it on the floor, it will take on a unique shape. Unlike metal chains that are made of identical links, proteins are made of 20 different amino acids that each have their own unique properties (different shapes, and attractive and repulsive forces, for example), and in combination, the amino acids exert forces on the chain to make it take on a specific shape, which we call a “fold.” The order in which the amino acids are linked determines the protein’s fold. There are many kinds of proteins that vary in the number and order of their amino acids.

To predict the shape that a particular protein adopts in nature, what we are really trying to do is find the fold with the lowest energy. The energy is determined by a number of factors. For example, some amino acids are attracted to each other so when they are close in space, their interaction provides a favorable contribution to the energy. Rosetta’s strategy for finding low energy shapes looks like this:

Start with a fully unfolded chain (like a metal chain with its ends pulled).
Move a part of the chain to create a new shape.
Calculate the energy of the new shape.
Accept or reject the move depending on the change in energy.
Repeat 2 through 4 until every part of the chain has been moved a lot of times.

We call this a trajectory. The end result of a trajectory is a predicted structure. Rosetta keeps track of the lowest energy shape found in each trajectory. Each trajectory is unique, because the attempted moves are determined by a random number. They do not always find the same low energy shape because there are so many possibilities.

A trajectory may consist of two stages. The first stage uses a simplified representation of amino acids which allows us to try many different possible shapes rapidly. This stage is regarded as a low resolution search and on the screen saver you will see the protein chain jumping around a lot. In the second stage, Rosetta uses a full representation of amino acids. This stage is refered to as “relaxation.” Instead of moving around a lot, the protein tries smaller changes in an attempt to move the amino acids to their correct arrangment. This stage is regarded as a high resolution search and on the screen saver, you will see the protein chain jiggle around a little. Rosetta can do the first stage in a few minutes on a modern computer. The second stage takes longer because of the increased complexity when considering the full representation (all atoms) of amino acids.

Your computer typically generates 5-20 of these trajectories (per work unit) and then sends us back the lowest energy shape seen in each one. We then look at all of the low energy shapes, generated by all of your computers, to find the very lowest ones. This becomes our prediction for the fold of that protein.

To join this project, download and install the BOINC software on which it runs. Then attach to the project. While you are at BOINC, look at some of the other projects to see what else might be of interest to you.

From Wyss: “From super to ultra-resolution microscopy”

Wyss Institute

Jul 5, 2016 [Just showed up in social media.]
PRESS CONTACTS

Wyss Institute for Biologically Inspired Engineering at Harvard University
Benjamin Boettner, benjamin.boettner@wyss.harvard.edu, +1 917-913-8051

MULTIMEDIA CONTACT

Wyss Institute for Biologically Inspired Engineering at Harvard University
Seth Kroll, seth.kroll@wyss.harvard.edu, +1 617-432-7758

The image shows how the Discrete Molecular Imaging (DMI) technology visualizes densely packed individual targets that are just 5 nanometer apart from each other in DNA origami structures (see schematics on the left). The image on the top right shows a DMI-generated super-resolution image of a clear pattern of individual signals. In the image on the bottom right, three different target species within the same origami structure have been visualized using Exchange-PAINT-enhanced DMI method. Credit: Wyss Institute at Harvard University.

Proteins mostly do not work in isolation but rather make up larger complexes like the molecular machines that enable cells to communicate with each other, move cargo around in their interiors or replicate their DNA. Our ability to observe and track each individual protein within these machines is crucial to our ultimate understanding of these processes. Yet, the advent of super-resolution microscopy that has allowed researchers to start visualizing closely positioned molecules or molecular complexes with 10-20 nanometer resolution is not powerful enough to distinguish individual molecular features within those densely packed complexes.

A team at Harvard’s Wyss Institute for Biologically Inspired Engineering led by Core Faculty member Peng Yin, Ph.D., has, for the first time, been able to tell apart features distanced only 5 nanometers from each other in a densely packed, single molecular structure and to achieve the so far highest resolution in optical microscopy. Reported on July 4 in a study in Nature Nanotechnology, the technology, also called “discrete molecular imaging” (DMI), enhances the team’s DNA nanotechnology-powered super-resolution microscopy platform with an integrated set of new imaging methods

Last year, the opportunity to enable researchers with inexpensive super-resolution microscopy using DNA-PAINT-based technologies led the Wyss Institute to launch its spin-off Ultivue Inc.

“The ultra-high resolution of DMI advances the DNA-PAINT platform one step further towards the vision of providing the ultimate view of biology. With this new power of resolution and the ability to focus on individual molecular features, DMI complements current structural biology methods like X-ray crystallography and cryo-electron microscopy. It opens up a way for researchers to study molecular conformations and heterogeneities in single multi-component complexes, and provides an easy, fast and multiplexed method for the structural analysis of many samples in parallel” said Peng Yin, who is also Professor of Systems Biology at Harvard Medical School.

DNA-PAINT technologies, developed by Yin and his team are based on the transient binding of two complementary short DNA strands, one being attached to the molecular target that the researchers aim to visualize and the other attached to a fluorescent dye. Repeated cycles of binding and unbinding create a very defined blinking behavior of the dye at the target site, which is highly programmable by the choice of DNA strands and has now been further exploited by the team’s current work to achieve ultra-high resolution imaging.

“By further harnessing key aspects underlying the blinking conditions in our DNA-PAINT-based technologies and developing a novel method that compensates for tiny but extremely disruptive movements of the microscope stage that carries the samples, we managed to additionally boost the potential beyond what has been possible so far in super-resolution microscopy,” said Mingjie Dai, who is the study’s first author and a Graduate Student working with Yin.

In addition, the study was co-authored by Ralf Jungmann, Ph.D., a former Postdoctoral Fellow on Yin’s team and now a Group Leader at the Max Planck Institute of Biochemistry at the Ludwig Maximilian University in Munich, Germany.

In this image the “Wyss!” name has been visualized in a DNA origami display with the so-far highest resolution possible in optical imaging using Discrete Molecular Imaging (DMI) technology. Credit: Wyss Institute at Harvard University.

The Wyss Institute’s scientists have benchmarked the ultra-high resolution of DMI using synthetic DNA nanostructures. Next, the researchers plan to apply the technology to actual biological complexes such as the protein complex that duplicates DNA in dividing cells or cell surface receptors binding their ligands.

“Peng Yin and his team have yet again broken through barriers never before possible by leveraging the power of programmable DNA, not for information storage, but create nanoscale ‘molecular instruments’ that carry out defined tasks and readout what they analyze. This new advancement to their DNA-powered super-resolution imaging platform is an amazing feat that has the potential to uncover the inner workings of cells at the single molecule level using conventional microscopes that are available in common biology laboratories,” said Donald Ingber, M.D., Ph.D., who is the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, and also Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

See the full article here .

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The Wyss (pronounced “Veese”) Institute for Biologically Inspired Engineering uses Nature’s design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world.

Working as an alliance among Harvard’s Schools of Medicine, Engineering, and Arts & Sciences, and in partnership with Beth Israel Deaconess Medical Center, Boston Children’s Hospital, Brigham and Women’s Hospital, Dana Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Tufts University, and Boston University, the Institute crosses disciplinary and institutional barriers to engage in high-risk research that leads to transformative technological breakthroughs.

From Technion: “Lethal Sequences”

Technion

31/07/2016
No writer credit found

Underrepresented sequences (URSs) inhibit protein translation and can be lethal. Top, The genetic information is transferred to the protein synthesis machine, the ribosome, by messenger RNA. Amino acids (colored circles) are added one by one and the newly synthesized protein is pushed out of the ribosome. On the left is a normal sequence. On the right is a sequence with the strong URS – CMYW, which slows translation and prevents ribosome recycling. Middle, left, proteins fold and assemble correctly. On the right, fewer full-length proteins are produced and truncated proteins are also produced due to the URS effect on the ribosome. Bottom, E. coli cells grown on plates in the absence (-) or presence (+) of the protein translation signal molecule IPTG. On the left, cells grow normally with or without IPTG. On the right, cells grow normally when the URS containing protein is not synthesized (no IPTG). When IPTG is added, the URS containing protein synthesis is initiated (+), ribosomes are inhibited, and fewer colonies of the bacteria grow. No image credit

Prof. Noam Adir, Dean of the Schulich Faculty of Chemistry.

A study from the lab of Prof. Noam Adir of the Schulich Faculty of Chemistry at Technion – Israel Institute of Technology: natural evolutionary processes prevent the presence of dangerous and potentially lethal molecular interactions by avoiding the presence of specific protein sequences in microorganisms. They found these sequences by a novel method – looking for what is missing in biological data sets. The group then experimentally showed that when these sequences are present in a protein, bacterial growth is indeed inhibited. The study was recently published in the Proceedings of the National Academy of Sciences, USA.

Evolution is an ongoing process, whereby those individuals of species that are the most fit for their environment have more offspring and thus out-compete less fit individuals. The individual’s fitness is a product of the quality of its cellular biochemistry, made possible by the thousands of enzymes that allow its physiology to perform all of the necessary chemical reactions that allow the cell to live. Deficiency in these molecular functions can lead to disease, loss of adaptability to environmental changes, or weakness against other organisms. The molecular machines that make life possible are large polymers made up of linear sequences of building blocks that contain different chemical functions: proteins, DNA, and RNA. Biological variety is a result of the evolutionary changes in these polymers, first and foremost the result of the astronomic number of possible permutations in the order of the 20 naturally occurring amino acid (AA) residues that are the building blocks of proteins. There are 8,000 possible sequences of three AAs, 160,000 sequences of four AAs, over 3 million sequences of five AAs and so on. Since proteins can contain between hundreds to thousands of AAs, the possibilities are endless.

The millions of different protein sequences found in all organisms determine the three-dimensional structures that give proteins the ability to function correctly. Proteins in cells can work alone or associate correctly with other cellular components, while avoiding incorrect and harmful associations with other components. Changes to the sequences naturally occur due to mutations (single site, or larger changes due to more dramatic sequence shuffling) of an organism’s DNA – the genetic material. Changes due to mutations can lead to new positive characteristics, or they may have negative consequences to the organism’s viability. A mutation that has a negative effect may prevent the organism from competing with other organisms in its environment, eventually leading to its demise. One could predict that over time, evolutionary pressure would work against the presence of organisms containing these internally lethal sequences and they would disappear.

Over the past few years, there has been a world-wide effort to obtain the entire DNA sequences (the entire genomes) of many organisms. These data have given us the ability to predict all of the possible protein sequences (the proteome) that might exist in organisms as simple as bacteria or as complicated as humans. Prof. Adir and his students, Dr. Sharon Penias-Navon and Ms. Tali Schwartzman, hypothesized that the huge amount of data made available by modern genomics would allow them to look for short sequences that occur less often than expected or are completely missing in the organism’s proteome. They developed a computer program that searched the many existing data sets to identify short sequences that are underrepresented (URSs). While they found that most of the sequences of three or four AAs indeed do exist at their expected frequency in the proteins of different organisms, URSs do exist. They used the program to search for URSs in the proteomes of many different organisms (especially pathogenic microorganisms) and found that different organisms have different URSs. Adir and Penias-Navon wanted to prove that these URSs are indeed harmful, and they hypothesized that protein synthesis (translation) by the ribosome is the function that URSs might harm.

They embedded bacterial URSs (identified in the proteome of the gut bacterium E. coli) comprised of three or four AAs in a normal protein sequence, and showed that no matter where they put the URS, protein translation was inhibited. They showed that these same E. coli URSs had no effect on protein translation in human cells, showing that the effect is species specific. They further showed that one four-AA URS was powerful enough to inhibit translation completely to the point where the growth of the bacterial cells was significantly reduced: these are indeed lethal sequences. Adir and Navon suggested that URSs could be used as highly specific anti-microbial agents, and a patent, together with the Technion, was submitted.

In order to obtain even more precise molecular details on the action of the URS, they initiated a collaboration with Prof. Joseph Puglisi and his student Dr. Guy Kornberg of Stanford University, who are experts in following protein translation in single ribosomes, thereby obtaining direct information on the translation reaction mechanism. Using these single molecule methods, the inhibitory effect of the existence of a URS on translation was confirmed. Their methods enabled a precise determination of the site of inhibition. They found that as soon as the URS AAs enter the entrance to the ribosomal nascent protein exit tunnel, translation is inhibited.

See the full article here .

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A science and technology research university, among the world’s top ten,
dedicated to the creation of knowledge and the development of human capital and leadership,
for the advancement of the State of Israel and all humanity.